Delamination resistant pharmaceutical glass containers containing active pharmaceutical ingredients

ABSTRACT

The present invention is based, at least in part, on the identification of a pharmaceutical container formed, at least in part, of a glass composition which exhibits a reduced propensity to delaminate, i.e., a reduced propensity to shed glass particulates. As a result, the presently claimed containers are particularly suited for storage of pharmaceutical compositions and, specifically, a pharmaceutical solution comprising a pharmaceutically active ingredient, for example, VELCADE (bortezomib), STELARA (Ustekinumab), SIMPONI (golimumab), siltuximab, and AMG 403 (fulranumab).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/815,678, filed Apr. 24, 2013, entitled “Delamination ResistantPharmaceutical Glass Containers Containing Active PharmaceuticalIngredients”, the entirety of which is hereby incorporated by referenceherein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 21, 2014, isnamed 122467-01202_SL.txt and is 14,214 bytes in size.

FIELD OF THE INVENTION

The present specification generally relates to pharmaceutical containersand, more specifically, to chemically and mechanically durablepharmaceutical containers that are delamination resistant and formed, atleast in part, of a glass composition.

BACKGROUND

The design of a packaged pharmaceutical composition generally seeks toprovide an active pharmaceutical ingredient (API) in a suitable packagethat is convenient to use, that maintains the stability of the API overprolonged storage, and that ultimately allows for the delivery ofefficacious, stable, active, nontoxic and nondegraded API.

Most packaged formulations are complex physico-chemical systems, throughwhich the API is subject to deterioration by a variety of chemical,physical, and microbial reactions. Interactions between drugs,adjuvants, containers, and/or closures may occur, which can lead to theinactivation, decomposition and/or degradation of the API.

Historically, glass has been used as the preferred material forpackaging pharmaceuticals because of its hermeticity, optical clarityand excellent chemical durability relative to other materials.Specifically, the glass used in pharmaceutical packaging must haveadequate chemical durability so as not to affect the stability of thepharmaceutical compositions contained therein. Glasses having suitablechemical durability include those glass compositions within the ASTMstandard ‘Type 1B’ glass compositions which have a proven history ofchemical durability.

However, use of glass for such applications is limited by the mechanicalperformance of the glass. Specifically, in the pharmaceutical industry,glass breakage is a safety concern for the end user as the brokenpackage and/or the contents of the package may injure the end user.Further, non-catastrophic breakage (i.e., when the glass cracks but doesnot break) may cause the contents to lose their sterility which, inturn, may result in costly product recalls.

One approach to improving the mechanical durability of the glass packageis to thermally temper the glass package. Thermal tempering strengthensglass by inducing a surface compressive stress during rapid coolingafter forming. This technique works well for glass articles with flatgeometries (such as windows), glass articles with thicknesses >2 mm, andglass compositions with high thermal expansion. However, pharmaceuticalglass packages typically have complex geometries (vial, tubular,ampoule, etc.), thin walls (˜1-1.5 mm), and are produced from lowexpansion glasses (30-55×10⁻⁷K⁻¹) making glass pharmaceutical packagesunsuitable for strengthening by thermal tempering.

Chemical tempering also strengthens glass by the introduction of surfacecompressive stress. The stress is introduced by submerging the articlein a molten salt bath. As ions from the glass are replaced by largerions from the molten salt, a compressive stress is induced in thesurface of the glass. The advantage of chemical tempering is that it canbe used on complex geometries, thin samples, and is relativelyinsensitive to the thermal expansion characteristics of the glasssubstrate. However, glass compositions which exhibit a moderatesusceptibility to chemical tempering generally exhibit poor chemicaldurability and vice-versa.

Finally, glass compositions commonly used in pharmaceutical packages,e.g., Type 1a and Type 1b glass, further suffer from a tendency for theinterior surfaces of the pharmaceutical package to shed glassparticulates or “delaminate” following exposure to pharmaceuticalsolutions. Such delamination often destabilizes the activepharmaceutical ingredient (API) present in the solution, therebyrendering the API therapeutically ineffective or unsuitable fortherapeutic use.

Delamination has caused the recall of multiple drug products over thelast few years (see, for example, Reynolds et al., (2011) BioProcessInternational 9(11) pp. 52-57). In response to the growing delaminationproblem, the U.S. Food and Drug Administration (FDA) has issued anadvisory indicating that the presence of glass particulate in injectabledrugs can pose a risk.

The advisory states that, “[t]here is potential for drugs administeredintravenously that contain these fragments to cause embolic, thromboticand other vascular events; and subcutaneously to the development offoreign body granuloma, local injections site reactions and increasedimmunogenicity.”

Accordingly, a recognized need exists for alternative glass containersfor packaging of pharmaceutical compositions which exhibit a reducedpropensity to delaminate.

SUMMARY

In one aspect, the present invention includes a delamination resistantpharmaceutical container including a glass composition. Thepharmaceutical container includes from about 70 mol. % to about 80 mol.% SiO₂; from about 3 mol. % to about 13 mol. % alkaline earth oxide; Xmol. % Al₂O₃; and Y mol. % alkali oxide. The alkali oxide includes Na₂Oin an amount greater than about 8 mol. %, a ratio of Y:X is greater than1, and the glass composition is free of boron and compounds of boron.The delamination resistant pharmaceutical container further includes anactive pharmaceutical ingredient.

In one or more embodiments, the SiO₂ is present in an amount less thanor equal to 78 mol. %. In some embodiments, an amount of the alkalineearth oxide is greater than or equal to about 4 mol. % and less than orequal to about 8 mol. %. In one or more embodiments, the alkaline earthoxide includes MgO and CaO and a ratio (CaO (mol. %)/(CaO (mol. %)+MgO(mol. %))) is less than or equal to 0.5. In one or more embodiments, thealkaline earth oxide includes from about 0.1 mol. % to less than orequal to about 1.0 mol. % CaO. In one or more embodiments, the alkalineearth oxide includes from about 3 mol. % to about 7 mol. % MgO. In oneor more embodiments, X is greater than or equal to about 2 mol. % andless than or equal to about 10 mol. %. In embodiments, the alkali oxideincludes greater than or equal to about 9 mol. % Na₂O and less than orequal to about 15 mol. % Na₂O. In some embodiments, the ratio of Y:X isless than or equal to 2. In one or more embodiments, the ratio of Y:X isgreater than or equal to 1.3 and less than or equal to 2.0. In one ormore embodiments, the alkali oxide further includes K₂O in an amountless than or equal to about 3 mol. %. In one or more embodiments, theglass composition is free of phosphorous and compounds of phosphorous.In one or more embodiments, the alkali oxide includes K₂O in an amountgreater than or equal to about 0.01 mol. % and less than or equal toabout 1.0 mol. %.

In another aspect, the invention includes a delamination resistantpharmaceutical container including a pharmaceutical composition. Thepharmaceutical container includes an active pharmaceutical ingredient,such that the pharmaceutical container includes a glass compositionincluding SiO₂ in a concentration greater than about 70 mol. %; alkalineearth oxide including MgO and CaO, wherein CaO is present in an amountgreater than or equal to about 0.1 mol. % and less than or equal toabout 1.0 mol. %, and a ratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %)))is less than or equal to 0.5; and Y mol. % alkali oxide, wherein thealkali oxide includes Na₂O in an amount greater than about 8 mol. %,such that the glass composition is free of boron and compounds of boron.

In another aspect, the invention includes a delamination resistantpharmaceutical container including a pharmaceutical compositionincluding an active pharmaceutical ingredient. The pharmaceuticalcontainer includes a glass composition including from about 72 mol. % toabout 78 mol. % SiO₂; from about 4 mol. % to about 8 mol. % alkalineearth oxide, wherein the alkaline earth oxide includes MgO and CaO and aratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) is less than or equalto 0.5; X mol. % Al₂O₃, such that X is greater than or equal to about 4mol. % and less than or equal to about 8 mol. %; and Y mol. % alkalioxide, such that the alkali oxide includes Na₂O in an amount greaterthan or equal to about 9 mol. % and less than or equal to about 15 mol.%, a ratio of Y:X is greater than 1, and the glass composition is freeof boron and compounds of boron.

In another aspect, the invention includes a delamination resistantpharmaceutical container including a pharmaceutical compositionincluding an active pharmaceutical ingredient. The pharmaceuticalcontainer includes a glass composition. The glass composition includesfrom about 70 mol. % to about 80 mol. % SiO₂; from about 3 mol. % toabout 13 mol. % alkaline earth oxide, such that the alkaline earth oxideincludes CaO in an amount greater than or equal to about 0.1 mol. % andless than or equal to about 1.0 mol. %, MgO, and a ratio (CaO (mol.%)/(CaO (mol. %)+MgO (mol. %))) is less than or equal to 0.5; X mol. %Al₂O₃, wherein X is greater than or equal to about 2 mol. % and lessthan or equal to about 10 mol. %; and Y mol. % alkali oxide, wherein thealkali oxide includes from about 0.01 mol. % to about 1.0 mol. % K₂O anda ratio of Y:X is greater than 1, and the glass composition is free ofboron and compounds of boron.

In one or more embodiments of any of the above aspects, thepharmaceutical composition includes VELCADE (bortezomib), STELARA(ustekinumab), SIMPONI (golimumab), siltuximab, and AMG 403(fulranumab).

In one aspect, the present invention includes a pharmaceuticalcomposition. The pharmaceutical composition includes VELCADE(bortezomib), STELARA (ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab) and a pharmaceutically acceptable excipient,such that the pharmaceutical composition is contained within a glasspharmaceutical container including an internal homogeneous layer.

In one or more embodiments, the pharmaceutical container has acompressive stress greater than or equal to 150 MPa. In one or moreembodiments, the pharmaceutical container has a compressive stressgreater than or equal to 250 MPa. In one or more embodiments, thepharmaceutical container includes a depth of layer greater than 30 μm.In one or more embodiments, the depth of layer is greater than 35 μm. Inone or more embodiments, the pharmaceutical composition demonstratesincreased stability, product integrity, or efficacy.

In one aspect, the present invention includes a pharmaceuticalcomposition. The pharmaceutical composition includes VELCADE(bortezomib), STELARA (ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab) and a pharmaceutically acceptable excipient,such that the pharmaceutical composition is contained within a glasspharmaceutical container including an internal homogeneous layer havinga compressive stress greater than or equal to 150.

In one or more embodiments, the pharmaceutical container includes adepth of layer greater than 10 μm. In one or more embodiments, thepharmaceutical container includes a depth of layer greater than 25 μm.In one or more embodiments, the pharmaceutical container includes adepth of layer greater than 30 μm. In one or more embodiments, thepharmaceutical container has a compressive stress greater than or equalto 300 MPa. In one or more embodiments, the pharmaceutical containerincludes increased stability, product integrity, or efficacy.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes VELCADE(bortezomib), STELARA (ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab) and a pharmaceutically acceptable excipient,such that the pharmaceutical composition is contained within a glasspharmaceutical container having a compressive stress greater than orequal to 150 MPa and a depth of layer greater than 10 μm, and such thatthe pharmaceutical composition demonstrates increased stability, productintegrity, or efficacy.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes VELCADE(bortezomib), STELARA (ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab) and a pharmaceutically acceptable excipient,such that the pharmaceutical composition is contained within a glasspharmaceutical container including a substantially homogeneous innerlayer, and such that the pharmaceutical composition demonstratesincreased stability, product integrity, or efficacy.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes VELCADE(bortezomib), STELARA (ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab) and a pharmaceutically acceptable excipient,such that the pharmaceutical composition is contained within a glasspharmaceutical container having a delamination factor of less than 3,wherein the pharmaceutical composition demonstrates increased stability,product integrity, or efficacy.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes VELCADE(bortezomib), STELARA (ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab) and a pharmaceutically acceptable excipient,such that the pharmaceutical composition is contained within a glasspharmaceutical container which is substantially free of boron, and suchthat the pharmaceutical composition demonstrates increased stability,product integrity, or efficacy.

In one or more embodiments, the glass pharmaceutical container has acompressive stress greater than or equal to 150 MPa and a depth of layergreater than 25 μm. In one or more embodiments, the glass pharmaceuticalcontainer has a compressive stress greater than or equal to 300 MPa anda depth of layer greater than 35 μm. In one or more embodiments, theglass pharmaceutical container includes a substantially homogeneousinner layer. In one or more embodiments, the glass pharmaceuticalcontainer has a compressive stress greater than or equal to 150 MPa anda depth of layer greater than 25 μm.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes VELCADE(bortezomib), STELARA (ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab) and a pharmaceutically acceptable excipient,such that the pharmaceutical composition is contained within a glasspharmaceutical container including a delamination factor of less than 3,and such that the pharmaceutical composition includes increasedstability, product integrity, or efficacy.

In one or more embodiments of any of the above aspects, the containerhas a compressive stress greater than or equal to 300 MPa. In one ormore embodiments, the container has a depth of layer greater than 25 μm.In one or more embodiments, the container has a depth of layer greaterthan 30 μm. In one or more embodiments, the container has a depth oflayer of at least 35 μm. In one or more embodiments, the container has acompressive stress greater than or equal to 300 MPa. In one or moreembodiments, the container has a compressive stress greater than orequal to 350 MPa.

Additional features and advantages will be set forth in the detaileddescription which follows, and in part will be readily apparent to thoseskilled in the art from that description or recognized by practicing theembodiments described herein, including the detailed description whichfollows, the claims, as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description describe various embodiments and areintended to provide an overview or framework for understanding thenature and character of the claimed subject matter. The accompanyingdrawings are included to provide a further understanding of the variousembodiments, and are incorporated into and constitute a part of thisspecification. The drawings illustrate the various embodiments describedherein, and together with the description serve to explain theprinciples and operations of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts the relationship between the ratio of alkalioxides to alumina (x-axis) and the strain point, annealing point, andsoftening point (y-axes) of inventive and comparative glasscompositions;

FIG. 2 graphically depicts the relationship between the ratio of alkalioxides to alumina (x-axis) and the maximum compressive stress and stresschange (y-axes) of inventive and comparative glass compositions;

FIG. 3 graphically depicts the relationship between the ratio of alkalioxides to alumina (x-axis) and hydrolytic resistance as determined fromthe ISO 720 standard (y-axis) of inventive and comparative glasscompositions;

FIG. 4 graphically depicts diffusivity D (y-axis) as a function of theratio (CaO/(CaO+MgO)) (x-axis) for inventive and comparative glasscompositions;

FIG. 5 graphically depicts the maximum compressive stress (y-axis) as afunction of the ratio (CaO/(CaO+MgO)) (x-axis) for inventive andcomparative glass compositions;

FIG. 6 graphically depicts diffusivity D (y-axis) as a function of theratio (B₂O₃/(R₂O—Al₂O₃)) (x-axis) for inventive and comparative glasscompositions; and

FIG. 7 graphically depicts the hydrolytic resistance as determined fromthe ISO 720 standard (y-axis) as a function of the ratio(B₂O₃/(R₂O—Al₂O₃)) (x-axis) for inventive and comparative glasscompositions.

DETAILED DESCRIPTION

The present invention is based, at least in part, on the identificationof a pharmaceutical container formed, at least in part, of a glasscomposition which exhibits a reduced propensity to delaminate, i.e., areduced propensity to shed glass particulates. As a result, thepresently claimed containers are particularly suited for storage,maintenance and/or delivery of therapeutically efficaciouspharmaceutical compositions and, in particular pharmaceutical solutionscomprising active pharmaceutical ingredients, for example, VELCADE(bortezomib), STELARA (Ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab).

Conventional glass containers or glass packages for containingpharmaceutical compositions are generally formed from glass compositionswhich are known to exhibit chemical durability and low thermalexpansion, such as alkali borosilicate glasses. While alkaliborosilicate glasses exhibit good chemical durability, containermanufacturers have sporadically observed silica-rich glass flakesdispersed in the solution contained in the glass containers as a resultof delamination, particularly when the solution has been stored indirect contact with the glass surface for long time periods (months toyears).

Delamination refers to a phenomenon in which glass particles arereleased from the surface of the glass following a series of leaching,corrosion, and/or weathering reactions. In general, the glass particlesare silica-rich flakes of glass which originate from the interiorsurface of the package as a result of the leaching of modifier ions intoa solution contained within the package. These flakes may generally befrom about 1 nm to 2 μm thick with a width greater than about 50 μm.

It has heretofore been hypothesized that delamination is due to thephase separation which occurs in alkali borosilicate glasses when theglass is exposed to the elevated temperatures used for reforming theglass into a container shape.

However, it is now believed that the delamination of the silica-richglass flakes from the interior surfaces of the glass containers is dueto the compositional characteristics of the glass container in itsas-formed condition. Specifically, the high silica content of alkaliborosilicate glasses increases the melting temperature of the glass.However, the alkali and borate components in the glass composition meltand/or vaporize at much lower temperatures. In particular, the boratespecies in the glass are highly volatile and evaporate from the surfaceof the glass at the high temperatures necessary to melt and form theglass.

Specifically, glass stock is reformed into glass containers at hightemperatures and in direct flames. The high temperatures cause thevolatile borate species to evaporate from portions of the surface of theglass. When this evaporation occurs within the interior volume of theglass container, the volatilized borate species are re-deposited inother areas of the glass causing compositional heterogeneities in theglass container, particularly with respect to the bulk of the glasscontainer. For example, as one end of a glass tube is closed to form thebottom or floor of the container, borate species may evaporate from thebottom portion of the tube and be re-deposited elsewhere in the tube. Asa result, the areas of the container exposed to higher temperatures havesilica-rich surfaces. Other areas of the container which are amenable toboron deposition may have a silica-rich surface with a boron-rich layerbelow the surface. Areas amenable to boron deposition are at atemperature greater than the anneal point of the glass composition butless than the hottest temperature the glass is subjected to duringreformation when the boron is incorporated into the surface of theglass. Solutions contained in the container may leach the boron from theboron-rich layer. As the boron-rich layer is leached from the glass, thesilica-rich surface begins to spall, shedding silica-rich flakes intothe solution.

Definitions

The term “softening point,” as used herein, refers to the temperature atwhich the viscosity of the glass composition is 1×10^(7.6) poise.

The term “annealing point,” as used herein, refers to the temperature atwhich the viscosity of the glass composition is 1×10¹³ poise.

The terms “strain point” and “T_(strain)” as used herein, refers to thetemperature at which the viscosity of the glass composition is 3×10¹⁴poise.

The term “CTE,” as used herein, refers to the coefficient of thermalexpansion of the glass composition over a temperature range from aboutroom temperature (RT) to about 300° C.

In the embodiments of the glass compositions described herein, theconcentrations of constituent components (e.g., SiO₂, Al₂O₃, and thelike) are specified in mole percent (mol. %) on an oxide basis, unlessotherwise specified.

The terms “free” and “substantially free,” when used to describe theconcentration and/or absence of a particular constituent component in aglass composition, means that the constituent component is notintentionally added to the glass composition. However, the glasscomposition may contain traces of the constituent component as acontaminant or tramp in amounts of less than 0.01 mol. %.

The term “chemical durability,” as used herein, refers to the ability ofthe glass composition to resist degradation upon exposure to specifiedchemical conditions. Specifically, the chemical durability of the glasscompositions described herein was assessed according to threeestablished material testing standards: DIN 12116 dated March 2001 andentitled “Testing of glass—Resistance to attack by a boiling aqueoussolution of hydrochloric acid—Method of test and classification”; ISO695:1991 entitled “Glass—Resistance to attack by a boiling aqueoussolution of mixed alkali—Method of test and classification”; and ISO720:1985 entitled “Glass—Hydrolytic resistance of glass grains at 121degrees C.—Method of test and classification.” The chemical durabilityof the glass may also be assessed according to ISO 719:1985“Glass—Hydrolytic resistance of glass grains at 98 degrees C.—Method oftest and classification,” in addition to the above referenced standards.The ISO 719 standard is a less rigorous version of the ISO 720 standardand, as such, it is believed that a glass which meets a specifiedclassification of the ISO 720 standard will also meet the correspondingclassification of the ISO 719 standard. The classifications associatedwith each standard are described in further detail herein.

Glass Compositions

Reference will now be made in detail to various embodiments ofpharmaceutical containers formed, at least in part, of glasscompositions which exhibit improved chemical and mechanical durabilityand, in particular, improved resistance to delamination. The glasscompositions may also be chemically strengthened thereby impartingincreased mechanical durability to the glass. The glass compositionsdescribed herein generally comprise silica (SiO₂), alumina (Al₂O₃),alkaline earth oxides (such as MgO and/or CaO), and alkali oxides (suchas Na₂O and/or K₂O) in amounts which impart chemical durability to theglass composition. Moreover, the alkali oxides present in the glasscompositions facilitate chemically strengthening the glass compositionsby ion exchange. Various embodiments of the glass compositions will bedescribed herein and further illustrated with reference to specificexamples.

The glass compositions described herein are alkali aluminosilicate glasscompositions which generally include a combination of SiO₂, Al₂O₃, atleast one alkaline earth oxide, and one or more alkali oxides, such asNa₂O and/or K₂O. In some embodiments, the glass compositions may be freefrom boron and compounds containing boron. The combination of thesecomponents enables a glass composition which is resistant to chemicaldegradation and is also suitable for chemical strengthening by ionexchange. In some embodiments the glass compositions may furthercomprise minor amounts of one or more additional oxides such as, forexample, SnO₂, ZrO₂, ZnO, TiO₂, As₂O₃ or the like. These components maybe added as fining agents and/or to further enhance the chemicaldurability of the glass composition.

In the embodiments of the glass compositions described herein SiO₂ isthe largest constituent of the composition and, as such, is the primaryconstituent of the resulting glass network. SiO₂ enhances the chemicaldurability of the glass and, in particular, the resistance of the glasscomposition to decomposition in acid and the resistance of the glasscomposition to decomposition in water. Accordingly, a high SiO₂concentration is generally desired. However, if the content of SiO₂ istoo high, the formability of the glass may be diminished as higherconcentrations of SiO₂ increase the difficulty of melting the glasswhich, in turn, adversely impacts the formability of the glass. In theembodiments described herein, the glass composition generally comprisesSiO₂ in an amount greater than or equal to 67 mol. % and less than orequal to about 80 mol. % or even less than or equal to 78 mol. %. Insome embodiments, the amount of SiO₂ in the glass composition may begreater than about 68 mol. %, greater than about 69 mol. % or evengreater than about 70 mol. %. In some other embodiments, the amount ofSiO₂ in the glass composition may be greater than 72 mol. %, greaterthan 73 mol. % or even greater than 74 mol. %. For example, in someembodiments, the glass composition may include from about 68 mol. % toabout 80 mol. % or even to about 78 mol. % SiO₂. In some otherembodiments the glass composition may include from about 69 mol. % toabout 80 mol. % or even to about 78 mol. % SiO₂. In some otherembodiments the glass composition may include from about 70 mol. % toabout 80 mol. % or even to about 78 mol. % SiO₂. In still otherembodiments, the glass composition comprises SiO₂ in an amount greaterthan or equal to 70 mol. % and less than or equal to 78 mol. %. In someembodiments, SiO₂ may be present in the glass composition in an amountfrom about 72 mol. % to about 78 mol. %. In some other embodiments, SiO₂may be present in the glass composition in an amount from about 73 mol.% to about 78 mol. %. In other embodiments, SiO₂ may be present in theglass composition in an amount from about 74 mol. % to about 78 mol. %.In still other embodiments, SiO₂ may be present in the glass compositionin an amount from about 70 mol. % to about 76 mol. %.

The glass compositions described herein further include Al₂O₃. Al₂O₃, inconjunction with alkali oxides present in the glass compositions such asNa₂O or the like, improves the susceptibility of the glass to ionexchange strengthening. In the embodiments described herein, Al₂O₃ ispresent in the glass compositions in X mol. % while the alkali oxidesare present in the glass composition in Y mol. %. The ratio Y:X in theglass compositions described herein is greater than 1 in order tofacilitate the aforementioned susceptibility to ion exchangestrengthening. Specifically, the diffusion coefficient or diffusivity Dof the glass composition relates to the rate at which alkali ionspenetrate into the glass surface during ion exchange. Glasses which havea ratio Y:X greater than about 0.9 or even greater than about 1 have agreater diffusivity than glasses which have a ratio Y:X less than 0.9.Glasses in which the alkali ions have a greater diffusivity can obtain agreater depth of layer for a given ion exchange time and ion exchangetemperature than glasses in which the alkali ions have a lowerdiffusivity. Moreover, as the ratio of Y:X increases, the strain point,anneal point, and softening point of the glass decrease, such that theglass is more readily formable. In addition, for a given ion exchangetime and ion exchange temperature, it has been found that compressivestresses induced in glasses which have a ratio Y:X greater than about0.9 and less than or equal to 2 are generally greater than thosegenerated in glasses in which the ratio Y:X is less than 0.9 or greaterthan 2. Accordingly, in some embodiments, the ratio of Y:X is greaterthan 0.9 or even greater than 1. In some embodiments, the ratio of Y:Xis greater than 0.9, or even greater than 1, and less than or equal toabout 2. In still other embodiments, the ratio of Y:X may be greaterthan or equal to about 1.3 and less than or equal to about 2.0 in orderto maximize the amount of compressive stress induced in the glass for aspecified ion exchange time and a specified ion exchange temperature.

However, if the amount of Al₂O₃ in the glass composition is too high,the resistance of the glass composition to acid attack is diminished.Accordingly, the glass compositions described herein generally includeAl₂O₃ in an amount greater than or equal to about 2 mol. % and less thanor equal to about 10 mol. %. In some embodiments, the amount of Al₂O₃ inthe glass composition is greater than or equal to about 4 mol. % andless than or equal to about 8 mol. %. In some other embodiments, theamount of Al₂O₃ in the glass composition is greater than or equal toabout 5 mol. % to less than or equal to about 7 mol. %. In some otherembodiments, the amount of Al₂O₃ in the glass composition is greaterthan or equal to about 6 mol. % to less than or equal to about 8 mol. %.In still other embodiments, the amount of Al₂O₃ in the glass compositionis greater than or equal to about 5 mol. % to less than or equal toabout 6 mol. %.

The glass compositions also include one or more alkali oxides such asNa₂O and/or K₂O. The alkali oxides facilitate the ion exchangeability ofthe glass composition and, as such, facilitate chemically strengtheningthe glass. The alkali oxide may include one or more of Na₂O and K₂O. Thealkali oxides are generally present in the glass composition in a totalconcentration of Y mol. %. In some embodiments described herein, Y maybe greater than about 2 mol. % and less than or equal to about 18 mol.%. In some other embodiments, Y may be greater than about 8 mol. %,greater than about 9 mol. %, greater than about 10 mol. % or evengreater than about 11 mol. %. For example, in some embodiments describedherein Y is greater than or equal to about 8 mol. % and less than orequal to about 18 mol. %. In still other embodiments, Y may be greaterthan or equal to about 9 mol. % and less than or equal to about 14 mol.%.

The ion exchangeability of the glass composition is primarily impartedto the glass composition by the amount of the alkali oxide Na₂Oinitially present in the glass composition prior to ion exchange.Accordingly, in the embodiments of the glass compositions describedherein, the alkali oxide present in the glass composition includes atleast Na₂O. Specifically, in order to achieve the desired compressivestrength and depth of layer in the glass composition upon ion exchangestrengthening, the glass compositions include Na₂O in an amount fromabout 2 mol. % to about 15 mol. % based on the molecular weight of theglass composition. In some embodiments the glass composition includes atleast about 8 mol. % of Na₂O based on the molecular weight of the glasscomposition. For example, the concentration of Na₂O may be greater than9 mol. %, greater than 10 mol. % or even greater than 11 mol. %. In someembodiments, the concentration of Na₂O may be greater than or equal to 9mol. % or even greater than or equal to 10 mol. %. For example, in someembodiments the glass composition may include Na₂O in an amount greaterthan or equal to about 9 mol. % and less than or equal to about 15 mol.% or even greater than or equal to about 9 mol. % and less than or equalto 13 mol. %.

As noted above, the alkali oxide in the glass composition may furtherinclude K₂O. The amount of K₂O present in the glass composition alsorelates to the ion exchangeability of the glass composition.Specifically, as the amount of K₂O present in the glass compositionincreases, the compressive stress obtainable through ion exchangedecreases as a result of the exchange of potassium and sodium ions.Accordingly, it is desirable to limit the amount of K₂O present in theglass composition. In some embodiments, the amount of K₂O is greaterthan or equal to 0 mol. % and less than or equal to 3 mol. %. In someembodiments, the amount of K₂O is less or equal to 2 mol. % or even lessthan or equal to 1.0 mol. %. In embodiments where the glass compositionincludes K₂O, the K₂O may be present in a concentration greater than orequal to about 0.01 mol. % and less than or equal to about 3.0 mol. % oreven greater than or equal to about 0.01 mol. % and less than or equalto about 2.0 mol. %. In some embodiments, the amount of K₂O present inthe glass composition is greater than or equal to about 0.01 mol. % andless than or equal to about 1.0 mol. %. Accordingly, it should beunderstood that K₂O need not be present in the glass composition.However, when K₂O is included in the glass composition, the amount ofK₂O is generally less than about 3 mol. % based on the molecular weightof the glass composition.

The alkaline earth oxides present in the composition improve themeltability of the glass batch materials and increase the chemicaldurability of the glass composition. In the glass compositions describedherein, the total mol. % of alkaline earth oxides present in the glasscompositions is generally less than the total mol. % of alkali oxidespresent in the glass compositions in order to improve the ionexchangeability of the glass composition. In the embodiments describedherein, the glass compositions generally include from about 3 mol. % toabout 13 mol. % of alkaline earth oxide. In some of these embodiments,the amount of alkaline earth oxide in the glass composition may be fromabout 4 mol. % to about 8 mol. % or even from about 4 mol. % to about 7mol. %.

The alkaline earth oxide in the glass composition may include MgO, CaO,SrO, BaO or combinations thereof. In some embodiments, the alkalineearth oxide includes MgO, CaO or combinations thereof. For example, inthe embodiments described herein the alkaline earth oxide includes MgO.MgO is present in the glass composition in an amount which is greaterthan or equal to about 3 mol. % and less than or equal to about 8 mol. %MgO. In some embodiments, MgO may be present in the glass composition inan amount which is greater than or equal to about 3 mol. % and less thanor equal to about 7 mol. % or even greater than or equal to 4 mol. % andless than or equal to about 7 mol. % by molecular weight of the glasscomposition.

In some embodiments, the alkaline earth oxide may further include CaO.In these embodiments CaO is present in the glass composition in anamount from about 0 mol. % to less than or equal to 6 mol. % bymolecular weight of the glass composition. For example, the amount ofCaO present in the glass composition may be less than or equal to 5 mol.%, less than or equal to 4 mol. %, less than or equal to 3 mol. %, oreven less than or equal to 2 mol. %. In some of these embodiments, CaOmay be present in the glass composition in an amount greater than orequal to about 0.1 mol. % and less than or equal to about 1.0 mol. %.For example, CaO may be present in the glass composition in an amountgreater than or equal to about 0.2 mol. % and less than or equal toabout 0.7 mol. % or even in an amount greater than or equal to about 0.3mol. % and less than or equal to about 0.6 mol. %.

In the embodiments described herein, the glass compositions aregenerally rich in MgO, (i.e., the concentration of MgO in the glasscomposition is greater than the concentration of the other alkalineearth oxides in the glass composition including, without limitation,CaO). Forming the glass composition such that the glass composition isMgO-rich improves the hydrolytic resistance of the resultant glass,particularly following ion exchange strengthening. Moreover, glasscompositions which are MgO-rich generally exhibit improved ion exchangeperformance relative to glass compositions which are rich in otheralkaline earth oxides. Specifically, glasses formed from MgO-rich glasscompositions generally have a greater diffusivity than glasscompositions which are rich in other alkaline earth oxides, such as CaO.The greater diffusivity enables the formation of a deeper depth of layerin the glass. MgO-rich glass compositions also enable a highercompressive stress to be achieved in the surface of the glass comparedto glass compositions which are rich in other alkaline earth oxides suchas CaO. In addition, it is generally understood that as the ion exchangeprocess proceeds and alkali ions penetrate more deeply into the glass,the maximum compressive stress achieved at the surface of the glass maydecrease with time. However, glasses formed from glass compositionswhich are MgO-rich exhibit a lower reduction in compressive stress thanglasses formed from glass compositions that are CaO-rich or rich inother alkaline earth oxides (i.e., glasses which are MgO-poor). Thus,MgO-rich glass compositions enable glasses which have higher compressivestress at the surface and greater depths of layer than glasses which arerich in other alkaline earth oxides.

In order to fully realize the benefits of MgO in the glass compositionsdescribed herein, it has been determined that the ratio of theconcentration of CaO to the sum of the concentration of CaO and theconcentration of MgO in mol. % (i.e., (CaO/(CaO+MgO)) should beminimized Specifically, it has been determined that (CaO/(CaO+MgO))should be less than or equal to 0.5. In some embodiments (CaO/(CaO+MgO))is less than or equal to 0.3 or even less than or equal to 0.2. In someother embodiments (CaO/(CaO+MgO)) may even be less than or equal to 0.1.

Boron oxide (B₂O₃) is a flux which may be added to glass compositions toreduce the viscosity at a given temperature (e.g., the strain, annealand softening temperatures) thereby improving the formability of theglass. However, it has been found that additions of boron significantlydecrease the diffusivity of sodium and potassium ions in the glasscomposition which, in turn, adversely impacts the ion exchangeperformance of the resultant glass. In particular, it has been foundthat additions of boron significantly increase the time required toachieve a given depth of layer relative to glass compositions which areboron free. Accordingly, in some embodiments described herein, theamount of boron added to the glass composition is minimized in order toimprove the ion exchange performance of the glass composition.

For example, it has been determined that the impact of boron on the ionexchange performance of a glass composition can be mitigated bycontrolling the ratio of the concentration of B₂O₃ to the differencebetween the total concentration of the alkali oxides (i.e., R₂O, where Ris the alkali metals) and alumina (i.e., B₂O₃ (mol. %)/(R₂O (mol.%)-Al₂O₃ (mol. %)). In particular, it has been determined that when theratio of B₂O₃/(R₂O—Al₂O₃) is greater than or equal to about 0 and lessthan about 0.3 or even less than about 0.2, the diffusivities of alkalioxides in the glass compositions are not diminished and, as such, theion exchange performance of the glass composition is maintained.Accordingly, in some embodiments, the ratio of B₂O₃/(R₂O—Al₂O₃) isgreater than 0 and less than or equal to 0.3. In some of theseembodiments, the ratio of B₂O₃/(R₂O—Al₂O₃) is greater than 0 and lessthan or equal to 0.2. In some embodiments, the ratio of B₂O₃/(R₂O—Al₂O₃)is greater than 0 and less than or equal to 0.15 or even less than orequal to 0.1. In some other embodiments, the ratio of B₂O₃/(R₂O—Al₂O₃)may be greater than 0 and less than or equal to 0.05. Maintaining theratio B₂O₃/(R₂O—Al₂O₃) to be less than or equal to 0.3 or even less thanor equal to 0.2 permits the inclusion of B₂O₃ to lower the strain point,anneal point and softening point of the glass composition without theB₂O₃ adversely impacting the ion exchange performance of the glass.

In the embodiments described herein, the concentration of B₂O₃ in theglass composition is generally less than or equal to about 4 mol. %,less than or equal to about 3 mol. %, less than or equal to about 2 mol.%, or even less than or equal to 1 mol. %. For example, in embodimentswhere B₂O₃ is present in the glass composition, the concentration ofB₂O₃ may be greater than about 0.01 mol. % and less than or equal to 4mol. %. In some of these embodiments, the concentration of B₂O₃ may begreater than about 0.01 mol. % and less than or equal to 3 mol. %. Insome embodiments, the B₂O₃ may be present in an amount greater than orequal to about 0.01 mol. % and less than or equal to 2 mol. %, or evenless than or equal to 1.5 mol. %. Alternatively, the B₂O₃ may be presentin an amount greater than or equal to about 1 mol. % and less than orequal to 4 mol. %, greater than or equal to about 1 mol. % and less thanor equal to 3 mol. % or even greater than or equal to about 1 mol. % andless than or equal to 2 mol. %. In some of these embodiments, theconcentration of B₂O₃ may be greater than or equal to about 0.1 mol. %and less than or equal to 1.0 mol. %.

While in some embodiments the concentration of B₂O₃ in the glasscomposition is minimized to improve the forming properties of the glasswithout detracting from the ion exchange performance of the glass, insome other embodiments the glass compositions are free from boron andcompounds of boron such as B₂O₃. Specifically, it has been determinedthat forming the glass composition without boron or compounds of boronimproves the ion exchangeability of the glass compositions by reducingthe process time and/or temperature required to achieve a specific valueof compressive stress and/or depth of layer.

In some embodiments of the glass compositions described herein, theglass compositions are free from phosphorous and compounds containingphosphorous including, without limitation, P₂O₅. Specifically, it hasbeen determined that formulating the glass composition withoutphosphorous or compounds of phosphorous increases the chemicaldurability of the glass composition.

In addition to the SiO₂, Al₂O₃, alkali oxides and alkaline earth oxides,the glass compositions described herein may optionally further compriseone or more fining agents such as, for example, SnO₂, As₂O₃, and/or Cl⁻(from NaCl or the like). When a fining agent is present in the glasscomposition, the fining agent may be present in an amount less than orequal to about 1 mol. % or even less than or equal to about 0.4 mol. %.For example, in some embodiments the glass composition may include SnO₂as a fining agent. In these embodiments SnO₂ may be present in the glasscomposition in an amount greater than about 0 mol. % and less than orequal to about 1 mol. % or even an amount greater than or equal to about0.01 mol. % and less than or equal to about 0.30 mol. %.

Moreover, the glass compositions described herein may comprise one ormore additional metal oxides to further improve the chemical durabilityof the glass composition. For example, the glass composition may furtherinclude ZnO, TiO₂, or ZrO₂, each of which further improves theresistance of the glass composition to chemical attack. In theseembodiments, the additional metal oxide may be present in an amountwhich is greater than or equal to about 0 mol. % and less than or equalto about 2 mol. %. For example, when the additional metal oxide is ZnO,the ZnO may be present in an amount greater than or equal to 1 mol. %and less than or equal to about 2 mol. %. When the additional metaloxide is ZrO₂ or TiO₂, the ZrO₂ or TiO₂ may be present in an amount lessthan or equal to about 1 mol. %.

Based on the foregoing, it should be understood that, in a firstexemplary embodiment, a glass composition may include: SiO₂ in aconcentration greater than about 70 mol. % and Y mol. % alkali oxide.The alkali oxide may include Na₂O in an amount greater than about 8 mol.%. The glass composition may be free of boron and compounds of boron.The concentration of SiO₂ in this glass composition may be greater thanor equal to about 72 mol. %, greater than 73 mol. % or even greater than74 mol. %. The glass composition of this first exemplary embodiment maybe free from phosphorous and compounds of phosphorous. The glasscomposition may also include X mol. % Al₂O₃. When Al₂O₃ is included, theratio of Y:X may be greater than 1. The concentration of Al₂O₃ may begreater than or equal to about 2 mol. % and less than or equal to about10 mol. %.

The glass composition of this first exemplary embodiment may alsoinclude alkaline earth oxide in an amount from about 3 mol. % to about13 mol. %. The alkaline earth oxide may include MgO and CaO. The CaO maybe present in an amount greater than or equal to about 0.1 mol. % andless than or equal to about 1.0 mol. %. A ratio (CaO (mol. %)/(CaO (mol.%)+MgO (mol. %))) may be less than or equal to 0.5.

In a second exemplary embodiment, a glass composition may include:greater than about 68 mol. % SiO₂; X mol. % Al₂O₃; Y mol. % alkalioxide; and B₂O₃. The alkali oxide may include Na₂O in an amount greaterthan about 8 mol %. A ratio (B₂O₃ (mol. %)/(Y mol. %−X mol. %) may begreater than 0 and less than 0.3. The concentration of SiO₂ in thisglass composition may be greater than or equal to about 72 mol. %,greater than 73 mol. % or even greater than 74 mol. %. The concentrationof Al₂O₃ may be greater than or equal to about 2 mol. % and less than orequal to about 10 mol. %. In this second exemplary embodiment, the ratioof Y:X may be greater than 1. When the ratio of Y:X is greater than 1,an upper bound of the ratio of Y:X may be less than or equal to 2. Theglass composition of this first exemplary embodiment may be free fromphosphorous and compounds of phosphorous.

The glass composition of this second exemplary embodiment may alsoinclude alkaline earth oxide. The alkaline earth oxide may include MgOand CaO. The CaO may be present in an amount greater than or equal toabout 0.1 mol. % and less than or equal to about 1.0 mol. %. A ratio(CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) may be less than or equal to0.5.

The concentration of B₂O₃ in this second exemplary embodiment may begreater than or equal to about 0.01 mol. % and less than or equal toabout 4 mol. %.

In a third exemplary embodiment, a glass article may have a type HgB1hydrolytic resistance according to ISO 719. The glass article mayinclude greater than about 8 mol. % Na₂O and less than about 4 mol. %B₂O₃. The glass article may further comprise X mol. % Al₂O₃ and Y mol. %alkali oxide. The ratio (B₂O₃ (mol. %)/(Y mol. %−X mol. %) may begreater than 0 and less than 0.3. The glass article of this thirdexemplary embodiment may further include a compressive stress layerhaving a surface compressive stress greater than or equal to about 250MPa. The glass article may also have at least a class S3 acid resistanceaccording to DIN 12116; at least a class A2 base resistance according toISO 695; and a type HgA1 hydrolytic resistance according to ISO 720.

In a fourth exemplary embodiment, a glass pharmaceutical package mayinclude SiO₂ in an amount greater than about 70 mol. %; X mol. % Al₂O₃;and Y mol. % alkali oxide. The alkali oxide may include Na₂O in anamount greater than about 8 mol. %. A ratio of a concentration of B₂O₃(mol. %) in the glass pharmaceutical package to (Y mol. %−X mol. %) maybe less than 0.3. The glass pharmaceutical package may also have a typeHGB1 hydrolytic resistance according to ISO 719. The concentration ofSiO₂ in the glass pharmaceutical package of this fourth exemplaryembodiment may be greater than or equal to 72 mol. % and less than orequal to about 78 mol. % or even greater than 74 mol. % and less than orequal to about 78 mol. %. The concentration of Al₂O₃ in the glasspharmaceutical may be greater than or equal to about 4 mol. % and lessthan or equal to about 8 mol. %. A ratio of Y:X may be greater than 1and less than 2.

The glass pharmaceutical package of this fourth exemplary embodiment mayalso include alkaline earth oxide in an amount from about 4 mol. % toabout 8 mol. %. The alkaline earth oxide may include MgO and CaO. TheCaO may be present in an amount greater than or equal to about 0.2 mol.% and less than or equal to about 0.7 mol. %. A ratio (CaO (mol. %)/(CaO(mol. %)+MgO (mol. %))) may be less than or equal to 0.5. The glasspharmaceutical package of this fourth exemplary embodiment may have atype HGA1 hydrolytic resistance according to ISO 720.

In a fifth exemplary embodiment, a glass composition may include fromabout 70 mol. % to about 80 mol. % SiO₂; from about 3 mol. % to about 13mol. % alkaline earth oxide; X mol. % Al₂O₃; and Y mol. % alkali oxide.The alkali oxide may include Na₂O in an amount greater than about 8 mol.%. A ratio of Y:X may be greater than 1. The glass composition may befree of boron and compounds of boron.

In a sixth exemplary embodiment, a glass composition may include fromabout 68 mol. % to about 80 mol. % SiO₂; from about 3 mol. % to about 13mol. % alkaline earth oxide; X mol. % Al₂O₃; and Y mol. % alkali oxide.The alkali oxide may include Na₂O in an amount greater than about 8 mol.%. The glass composition of this sixth exemplary embodiment may alsoinclude B₂O₃. A ratio (B₂O₃ (mol. %)/(Y mol. %−X mol. %) may be greaterthan 0 and less than 0.3. A ratio of Y:X may be greater than 1.

In a seventh exemplary embodiment, a glass composition may include fromabout 70 mol. % to about 80 mol. % SiO₂; from about 3 mol. % to about 13mol. % alkaline earth oxide; X mol. % Al₂O₃; and Y mol. % alkali oxide.The amount of Al₂O₃ in the glass composition may be greater than orequal to about 2 mol. % and less than or equal to about 10 mol. %. Thealkaline earth oxide may include CaO in an amount greater than or equalto about 0.1 mol. % and less than or equal to about 1.0 mol. %. Thealkali oxide may include from about 0.01 mol. % to about 1.0 mol. % K₂O.A ratio of Y:X may be greater than 1. The glass composition may be freeof boron and compounds of boron. The glass composition may be amenableto strengthening by ion exchange.

In a seventh exemplary embodiment, a glass composition may include SiO₂in an amount greater than about 70 mol. % and less than or equal toabout 80 mol. %; X mol. % Al₂O₃; and Y mol. % alkali oxide. The alkalioxide may include Na₂O in an amount greater than about 8 mol. %. A ratioof a concentration of B₂O₃ (mol. %) in the glass pharmaceutical packageto (Y mol. %−X mol. %) may be less than 0.3. A ratio of Y:X may begreater than 1.

In an eighth exemplary embodiment, a glass composition may include fromabout 72 mol. % to about 78 mol. % SiO₂; from about 4 mol. % to about 8mol. % alkaline earth oxide; X mol. % Al₂O₃, wherein X is greater thanor equal to about 4 mol. % and less than or equal to about 8 mol. %; andY mol. % alkali oxide, wherein the alkali oxide comprises Na₂O in anamount greater than or equal to about 9 mol. % and less than or equal toabout 15 mol. %. A ratio of a concentration of B₂O₃ (mol. %) in theglass pharmaceutical package to (Y mol. %−X mol. %) is less than 0.3. Aratio of Y:X may be greater than 1.

In a ninth exemplary embodiment, a pharmaceutical package for containinga pharmaceutical composition may include from about 70 mol. % to about78 mol. % SiO₂; from about 3 mol. % to about 13 mol. % alkaline earthoxide; X mol. % Al₂O₃, wherein X is greater than or equal to 2 mol. %and less than or equal to about 10 mol. %; and Y mol. % alkali oxide,wherein the alkali oxide comprises Na₂O in an amount greater than about8 mol. %. The alkaline earth oxide may include CaO in an amount lessthan or equal to about 6.0 mol. %. A ratio of Y:X may be greater thanabout 1. The package may be free of boron and compounds of boron and mayinclude a compressive stress layer with a compressive stress greaterthan or equal to about 250 MPa and a depth of layer greater than orequal to about 10 μm.

In a tenth exemplary embodiment, a glass article may be formed from aglass composition comprising from about 70 mol. % to about 78 mol. %SiO₂; alkaline earth oxide, wherein the alkaline earth oxide comprisesMgO and CaO and a ratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) isless than or equal to 0.5; X mol. % Al₂O₃, wherein X is from about 2mol. % to about 10 mol. %; and Y mol. % alkali oxide, wherein the alkalioxide comprises Na₂O in an amount greater than about 8 mol. % and aratio of Y:X is greater than 1. The glass article may be ion exchangestrengthened with a compressive stress greater than or equal to 250 MPaand a depth of layer greater than or equal to 10 μm. The glass articlemay have a type HgA1 hydrolytic resistance according to ISO 720.

As noted above, the presence of alkali oxides in the glass compositionfacilitates chemically strengthening the glass by ion exchange.Specifically, alkali ions, such as potassium ions, sodium ions and thelike, are sufficiently mobile in the glass to facilitate ion exchange.In some embodiments, the glass composition is ion exchangeable to form acompressive stress layer having a depth of layer greater than or equalto 10 μm. In some embodiments, the depth of layer may be greater than orequal to about 25 μm or even greater than or equal to about 50 μm. Insome other embodiments, the depth of the layer may be greater than orequal to 75 μm or even greater than or equal to 100 μm. In still otherembodiments, the depth of layer may be greater than or equal to 10 μmand less than or equal to about 100 μm. The associated surfacecompressive stress may be greater than or equal to about 250 MPa,greater than or equal to 300 MPa or even greater than or equal to about350 MPa after the glass composition is treated in a salt bath of 100%molten KNO₃ at a temperature of 350° C. to 500° C. for a time period ofless than about 30 hours or even about less than 20 hours.

The glass articles formed from the glass compositions described hereinmay have a hydrolytic resistance of HGB2 or even HGB1 under ISO 719and/or a hydrolytic resistance of HGA2 or even HGA1 under ISO 720 (asdescribed further herein) in addition to having improved mechanicalcharacteristics due to ion exchange strengthening. In some embodimentsdescribed herein the glass articles may have compressive stresses whichextend from the surface into the glass article to a depth of layergreater than or equal to 10 μm, greater than or equal to 15 μm, greaterthan or equal to 20 μm, greater than or equal to 25 μm, greater than orequal to 30 μm or even greater than or equal to 35 μm. In someembodiments, the depth of layer may be greater than or equal to 40 μm oreven greater than or equal to 50 μm. The surface compressive stress ofthe glass article may be greater than or equal to 150 MPa, greater thanor equal to 200 MPa, greater than or equal to 250 MPa, greater than orequal to 350 MPa, or even greater than or equal to 400 MPa.

In one embodiment, the glass pharmaceutical container has a compressivestress greater than or equal to 150 MPa and a depth of layer greaterthan or equal to 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μmor 50 μm. In a particular embodiment, the glass pharmaceutical containerhas a compressive stress greater than or equal to 150 MPa and a depth oflayer greater than or equal to 10 μm. In a particular embodiment, theglass pharmaceutical container has a compressive stress greater than orequal to 150 MPa and a depth of layer greater than or equal to 25 μm. Ina particular embodiment, the glass pharmaceutical container has acompressive stress greater than or equal to 150 MPa and a depth of layergreater than or equal to 30 μm.

In one embodiment, the glass pharmaceutical container has a compressivestress greater than or equal to 300 MPa and a depth of layer greaterthan or equal to 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μmor 50 μm. In a particular embodiment, the glass pharmaceutical containerhas a compressive stress greater than or equal to 300 MPa and a depth oflayer greater than or equal to 25 μm. In yet another embodiment, theglass pharmaceutical container has a compressive stress greater than orequal to 300 MPa and a depth of layer greater than or equal to 30 μm. Inyet another embodiment, the glass pharmaceutical container has acompressive stress greater than or equal to 300 MPa and a depth of layergreater than or equal to 35 μm.

The glass compositions described herein facilitate achieving theaforementioned depths of layer and surface compressive stresses morerapidly and/or at lower temperatures than conventional glasscompositions due to the enhanced alkali ion diffusivity of the glasscompositions as described hereinabove. For example, the depths of layer(i.e., greater than or equal to 25 μm) and the compressive stresses(i.e., greater than or equal to 250 MPa) may be achieved by ionexchanging the glass article in a molten salt bath of 100% KNO₃ (or amixed salt bath of KNO₃ and NaNO₃) for a time period of less than orequal to 5 hours or even less than or equal to 4.5 hours. In someembodiments, these depths of layer and compressive stresses may beachieved by ion exchanging the glass article in a molten salt bath of100% KNO₃ (or a mixed salt bath of KNO₃ and NaNO₃) for a time period ofless than or equal to 4 hours or even less than or equal to 3.5 hours.Moreover, these depths of layers and compressive stresses may beachieved by ion exchanging the glass articles in a molten salt bath of100% KNO3 (or a mixed salt bath of KNO₃ and NaNO₃) at a temperature lessthan or equal to 500° C. or even less than or equal to 450° C. In someembodiments, these depths of layers and compressive stresses may beachieved by ion exchanging the glass articles in a molten salt bath of100% KNO3 (or a mixed salt bath of KNO₃ and NaNO₃) at a temperature lessthan or equal to 400° C. or even less than or equal to 350° C.

These improved ion exchange characteristics can be achieved when theglass composition has a threshold diffusivity of greater than about 16μm²/hr or even greater than or equal to 20 μm²/hr at 450° C. In someembodiments, the threshold diffusivity may be greater than or equal toabout 25 m²/hr or even 30 μm²/hr at 450° C. In some other embodiments,the threshold diffusivity may be greater than or equal to about 35 m²/hror even 40 μm²/hr at 450° C. In still other embodiments, the thresholddiffusivity may be greater than or equal to about 45 μm²/hr or even 50μm²/hr at 450° C.

The glass compositions described herein may generally have a strainpoint greater than or equal to about 525° C. and less than or equal toabout 650° C. The glasses may also have an anneal point greater than orequal to about 560° C. and less than or equal to about 725° C. and asoftening point greater than or equal to about 750° C. and less than orequal to about 960° C.

In the embodiments described herein the glass compositions have a CTE ofless than about 70×10⁻⁷K⁻¹ or even less than about 60×10⁻⁷K⁻¹. Theselower CTE values improve the survivability of the glass to thermalcycling or thermal stress conditions relative to glass compositions withhigher CTEs.

Further, as noted hereinabove, the glass compositions are chemicallydurable and resistant to degradation as determined by the DIN 12116standard, the ISO 695 standard, and the ISO 720 standard.

Specifically, the DIN 12116 standard is a measure of the resistance ofthe glass to decomposition when placed in an acidic solution. In brief,the DIN 12116 standard utilizes a polished glass sample of a knownsurface area which is weighed and then positioned in contact with aproportional amount of boiling 6M hydrochloric acid for 6 hours. Thesample is then removed from the solution, dried and weighed again. Theglass mass lost during exposure to the acidic solution is a measure ofthe acid durability of the sample with smaller numbers indicative ofgreater durability. The results of the test are reported in units ofhalf-mass per surface area, specifically mg/dm². The DIN 12116 standardis broken into individual classes. Class S1 indicates weight losses ofup to 0.7 mg/dm²; Class S2 indicates weight losses from 0.7 mg/dm² up to1.5 mg/dm²; Class S3 indicates weight losses from 1.5 mg/dm² up to 15mg/dm²; and Class S4 indicates weight losses of more than 15 mg/dm².

The ISO 695 standard is a measure of the resistance of the glass todecomposition when placed in a basic solution. In brief, the ISO 695standard utilizes a polished glass sample which is weighed and thenplaced in a solution of boiling 1M NaOH+0.5M Na₂CO₃ for 3 hours. Thesample is then removed from the solution, dried and weighed again. Theglass mass lost during exposure to the basic solution is a measure ofthe base durability of the sample with smaller numbers indicative ofgreater durability. As with the DIN 12116 standard, the results of theISO 695 standard are reported in units of mass per surface area,specifically mg/dm². The ISO 695 standard is broken into individualclasses. Class A1 indicates weight losses of up to 75 mg/dm²; Class A2indicates weight losses from 75 mg/dm² up to 175 mg/dm²; and Class A3indicates weight losses of more than 175 mg/dm².

The ISO 720 standard is a measure of the resistance of the glass todegradation in purified, CO₂-free water. In brief, the ISO 720 standardprotocol utilizes crushed glass grains which are placed in contact withthe purified, CO₂-free water under autoclave conditions (121° C., 2 atm)for 30 minutes. The solution is then titrated colorimetrically withdilute HCl to neutral pH. The amount of HCl required to titrate to aneutral solution is then converted to an equivalent of Na₂O extractedfrom the glass and reported in μg Na₂O per weight of glass with smallervalues indicative of greater durability. The ISO 720 standard is brokeninto individual types. Type HGA1 is indicative of up to 62 μg extractedequivalent of Na₂O per gram of glass tested; Type HGA2 is indicative ofmore than 62 μg and up to 527 μg extracted equivalent of Na₂O per gramof glass tested; and Type HGA3 is indicative of more than 527 μg and upto 930 μg extracted equivalent of Na₂O per gram of glass tested.

The ISO 719 standard is a measure of the resistance of the glass todegradation in purified, CO₂-free water. In brief, the ISO 719 standardprotocol utilizes crushed glass grains which are placed in contact withthe purified, CO₂-free water at a temperature of 98° C. at 1 atmospherefor 30 minutes. The solution is then titrated colorimetrically withdilute HCl to neutral pH. The amount of HCl required to titrate to aneutral solution is then converted to an equivalent of Na₂O extractedfrom the glass and reported in μg Na₂O per weight of glass with smallervalues indicative of greater durability. The ISO 719 standard is brokeninto individual types. The ISO 719 standard is broken into individualtypes. Type HGB1 is indicative of up to 31 μg extracted equivalent ofNa₂O; Type HGB2 is indicative of more than 31 μg and up to 62 μgextracted equivalent of Na₂O; Type HGB3 is indicative of more than 62 μgand up to 264 μg extracted equivalent of Na₂O; Type HGB4 is indicativeof more than 264 μg and up to 620 μg extracted equivalent of Na₂O; andType HGB5 is indicative of more than 620 μg and up to 1085 μg extractedequivalent of Na₂O. The glass compositions described herein have an ISO719 hydrolytic resistance of type HGB2 or better with some embodimentshaving a type HGB1 hydrolytic resistance.

The glass compositions described herein have an acid resistance of atleast class S3 according to DIN 12116 both before and after ion exchangestrengthening with some embodiments having an acid resistance of atleast class S2 or even class 51 following ion exchange strengthening. Insome other embodiments, the glass compositions may have an acidresistance of at least class S2 both before and after ion exchangestrengthening with some embodiments having an acid resistance of class51 following ion exchange strengthening. Further, the glass compositionsdescribed herein have a base resistance according to ISO 695 of at leastclass A2 before and after ion exchange strengthening with someembodiments having a class A1 base resistance at least after ionexchange strengthening. The glass compositions described herein alsohave an ISO 720 type HGA2 hydrolytic resistance both before and afterion exchange strengthening with some embodiments having a type HGA1hydrolytic resistance after ion exchange strengthening and some otherembodiments having a type HGA1 hydrolytic resistance both before andafter ion exchange strengthening. The glass compositions describedherein have an ISO 719 hydrolytic resistance of type HGB2 or better withsome embodiments having a type HGB1 hydrolytic resistance. It should beunderstood that, when referring to the above referenced classificationsaccording to DIN 12116, ISO 695, ISO 720 and ISO 719, a glasscomposition or glass article which has “at least” a specifiedclassification means that the performance of the glass composition is asgood as or better than the specified classification. For example, aglass article which has a DIN 12116 acid resistance of “at least classS2” may have a DIN 12116 classification of either S1 or S2.

The glass compositions described herein are formed by mixing a batch ofglass raw materials (e.g., powders of SiO₂, Al₂O₃, alkali oxides,alkaline earth oxides and the like) such that the batch of glass rawmaterials has the desired composition. Thereafter, the batch of glassraw materials is heated to form a molten glass composition which issubsequently cooled and solidified to form the glass composition. Duringsolidification (i.e., when the glass composition is plasticallydeformable) the glass composition may be shaped using standard formingtechniques to shape the glass composition into a desired final form.Alternatively, the glass article may be shaped into a stock form, suchas a sheet, tube or the like, and subsequently reheated and formed intothe desired final form.

In order to assess the long-term resistance of the glass container todelamination, an accelerated delamination test was utilized. The test isperformed on glass containers after the containers have beenion-exchange strengthened. The test consisted of washing the glasscontainer at room temperature for 1 minute and depyrogenating thecontainer at about 320° C. for 1 hour. Thereafter a solution of 20 mMglycine with a pH of 10 in water is placed in the glass container to80-90% fill, the glass container is closed, and rapidly heated to 100°C. and then heated from 100° C. to 121° C. at a ramp rate of 1 deg/minat a pressure of 2 atmospheres. The glass container and solution areheld at this temperature for 60 minutes, cooled to room temperature at arate of 0.5 deg./min and the heating cycle and hold are repeated. Theglass container is then heated to 50° C. and held for two days forelevated temperature conditioning. After heating, the glass container isdropped from a distance of at least 18″ onto a firm surface, such as alaminated tile floor, to dislodge any flakes or particles that areweakly adhered to the inner surface of the glass container.

Thereafter, the solution contained in the glass container is analyzed todetermine the number of glass particles present per liter of solution.Specifically, the solution from the glass container is directly pouredonto the center of a Millipore Isopore Membrane filter (Millipore#ATTP02500 held in an assembly with parts #AP1002500 and #M000025A0)attached to vacuum suction to draw the solution through the filterwithin 10-15 seconds. Particulate flakes are then counted bydifferential interference contrast microscopy (DIC) in the reflectionmode as described in “Differential interference contrast (DIC)microscopy and modulation contrast microscopy” from Fundamentals oflight microscopy and digital imaging. New York: Wiley-Liss, pp 153-168.The field of view is set to approximately 1.5 mm×1.5 mm and particleslarger than 50 microns are counted manually. There are 9 suchmeasurements made in the center of each filter membrane in a 3×3 patternwith no overlap between images. A minimum of 100 mL of solution istested. As such, the solution from a plurality of small containers maybe pooled to bring the total amount of solution to 100 mL. If thecontainers contain more than 10 mL of solution, the entire amount ofsolution from the container is examined for the presence of particles.For containers having a volume greater than 10 mL containers, the testis repeated for a trial of 10 containers formed from the same glasscomposition under the same processing conditions and the result of theparticle count is averaged for the 10 containers to determine an averageparticle count. Alternatively, in the case of small containers, the testis repeated for a trial of 10 sets of 10 mL of solution, each of whichis analyzed and the particle count averaged over the 10 sets todetermine an average particle count. Averaging the particle count overmultiple containers accounts for potential variations in thedelamination behavior of individual containers.

It should be understood that the aforementioned test is used to identifyparticles which are shed from the interior wall(s) of the glasscontainer due to delamination and not tramp particles present in thecontainer from forming processes or particles which precipitate from thesolution enclosed in the glass container as a result of reactionsbetween the solution and the glass. Specifically, delamination particlesmay be differentiated from tramp glass particles due based on the aspectratio of the particle (i.e., the ratio of the width of the particle tothe thickness of the particle). Delamination produces particulate flakesor lamellae which are irregularly shaped and are typically >50 μm indiameter but often >200 μm. The thickness of the flakes is usuallygreater than about 100 nm and may be as large as about 1 μm. Thus, theminimum aspect ratio of the flakes is typically >50. The aspect ratiomay be greater than 100 and sometimes greater than 1000. Particlesresulting from delamination processes generally have an aspect ratiowhich is generally greater than about 50. In contrast, tramp glassparticles will generally have a low aspect ratio which is less thanabout 3. Accordingly, particles resulting from delamination may bedifferentiated from tramp particles based on aspect ratio duringobservation with the microscope. Validation results can be accomplishedby evaluating the heel region of the tested containers. Uponobservation, evidence of skin corrosion/pitting/flake removal, asdescribed in “Nondestructive Detection of Glass Vial Inner SurfaceMorphology with Differential Interference Contrast Microscopy” fromJournal of Pharmaceutical Sciences 101(4), 2012, pages 1378-1384, isnoted.

In the embodiments described herein, glass containers which average lessthan 3 glass particles with a minimum width of 50 μm and an aspect ratioof greater than 50 per trial following accelerated delamination testingare considered to have a delamination factor of 3. In the embodimentsdescribed herein, glass containers which average less than 2 glassparticles with a minimum width of 50 μm and an aspect ratio of greaterthan 50 per trial following accelerated delamination testing areconsidered to have a delamination factor of 2. In the embodimentsdescribed herein, glass containers which average less than 1 glassparticle with a minimum width of 50 μm and an aspect ratio of greaterthan 50 per trial following accelerated delamination testing areconsidered to have a delamination factor of 1. In the embodimentsdescribed herein, glass containers which have 0 glass particles with aminimum width of 50 μm and an aspect ratio of greater than 50 per trialfollowing accelerated delamination testing are considered to have adelamination factor of 0. Accordingly, it should be understood that thelower the delamination factor, the better the resistance of the glasscontainer to delamination. In the embodiments described herein, theglass containers have a delamination factor of 3 or lower (i.e., adelamination factor of 3, 2, 1 or 0).

Pharmaceutical Containers

In view of the chemical durability of the glass composition of thepresent invention, the glass compositions described herein areparticularly well suited for use in designing pharmaceutical containersfor storing, maintaining and/or delivering pharmaceutical compositions,such as liquids, solutions, powders, e.g., lyophilized powders, solidsand the like. As used herein, the term “pharmaceutical container” refersto a composition designed to store, maintain and/or deliver apharmaceutical composition. The pharmaceutical containers, as describedherein, are formed, at least in part, of the delamination resistantglass compositions described above. Pharmaceutical containers of thepresent invention include, but are not limited to, Vacutainers™cartridges, syringes, ampoules, bottles, flasks, phials, tubes, beakers,vials, injection pens or the like. In a particular embodiment, thepharmaceutical container is a vial. In a particular embodiment, thepharmaceutical container is an ampoule. In a particular embodiment, thepharmaceutical container is an injection pen. In a particularembodiment, the pharmaceutical container is a tube. In a particularembodiment, the pharmaceutical container is a bottle. In a particularembodiment, the pharmaceutical container is a syringe.

Moreover, the ability to chemically strengthen the glass compositionsthrough ion exchange can be utilized to improve the mechanicaldurability of pharmaceutical containers formed from the glasscomposition. Accordingly, it should be understood that, in at least oneembodiment, the glass compositions are incorporated in a pharmaceuticalcontainer in order to improve the chemical durability and/or themechanical durability of the pharmaceutical container.

Pharmaceutical Compositions

In various embodiments, the pharmaceutical container further includes apharmaceutical composition comprising an active pharmaceuticalingredient (API). As used herein, the term “pharmaceutical composition”refers to a composition comprising an active pharmaceutical ingredientto be delivered to a subject, for example, for therapeutic,prophylactic, diagnostic, preventative or prognostic effect. In certainembodiments, the pharmaceutical composition comprises the activepharmaceutical ingredient and a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it may be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the activepharmaceutical agent.

As used herein, the term “active pharmaceutical ingredient” or “API”refers a substance in a pharmaceutical composition that provides adesired effect, for example, a therapeutic, prophylactic, diagnostic,preventative or prognostic effect. In various embodiments, the activepharmaceutical ingredient can be any of a variety of substances known inthe art, for example, a small molecule, a polypeptide mimetic, abiologic, an antisense RNA, a small interfering RNA (siRNA), etc.

For example, in a particular embodiment, the active pharmaceuticalingredient may be a small molecule. As used herein, the term “smallmolecule” includes any chemical or other moiety, other than polypeptidesand nucleic acids, that can act to affect biological processes. Smallmolecules can include any number of therapeutic agents presently knownand used, or that can be synthesized from a library of such moleculesfor the purpose of screening for biological function(s). Small moleculesare distinguished from macromolecules by size. The small molecules ofthe present invention usually have a molecular weight less than about5,000 daltons (Da), preferably less than about 2,500 Da, more preferablyless than 1,000 Da, most preferably less than about 500 Da.

Small molecules include, without limitation, organic compounds,peptidomimetics and conjugates thereof. As used herein, the term“organic compound” refers to any carbon-based compound other thanmacromolecules such as nucleic acids and polypeptides. In addition tocarbon, organic compounds may contain calcium, chlorine, fluorine,copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and otherelements. An organic compound may be in an aromatic or aliphatic form.Non-limiting examples of organic compounds include acetones, alcohols,anilines, carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, nucleosides, nucleotides, lipids,retinoids, steroids, proteoglycans, ketones, aldehydes, saturated,unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters,ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds,imidizoles, and phenols. An organic compound as used herein alsoincludes nitrated organic compounds and halogenated (e.g., chlorinated)organic compounds.

In another embodiment, the active pharmaceutical ingredient may be apolypeptide mimetic (“peptidomimetic”). As used herein, the term“polypeptide mimetic” is a molecule that mimics the biological activityof a polypeptide, but that is not peptidic in chemical nature. While, incertain embodiments, a peptidomimetic is a molecule that contains nopeptide bonds (that is, amide bonds between amino acids), the termpeptidomimetic may include molecules that are not completely peptidic incharacter, such as pseudo-peptides, semi-peptides, and peptoids.

In other embodiments, the active pharmaceutical ingredient may be abiologic. As used herein, the term “biologic” includes products createdby biologic processes instead of by chemical synthesis. Non-limitingexamples of a “biologic” include proteins, antibodies, antibody likemolecules, vaccines, blood, blood components, and partially purifiedproducts from tissues.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein. In the present invention, these terms mean alinked sequence of amino acids, which may be natural, synthetic, or amodification or combination of natural and synthetic. The term includesantibodies, antibody mimetics, domain antibodies, lipocalins, andtargeted proteases. The term also includes vaccines containing a peptideor peptide fragment intended to raise antibodies against the peptide orpeptide fragment.

“Antibody” as used herein includes an antibody of classes IgG, IgM, IgA,IgD, or IgE, or fragments or derivatives thereof, including Fab,F(ab′)2, Fd, and single chain antibodies, diabodies, bispecificantibodies, and bifunctional antibodies. The antibody may be amonoclonal antibody, polyclonal antibody, affinity purified antibody, ormixtures thereof, which exhibits sufficient binding specificity to adesired epitope or a sequence derived therefrom. The antibody may alsobe a chimeric antibody. The antibody may be derivatized by theattachment of one or more chemical, peptide, or polypeptide moietiesknown in the art. The antibody may be conjugated with a chemical moiety.The antibody may be a human or humanized antibody.

Other antibody-like molecules are also within the scope of the presentinvention. Such antibody-like molecules include, e.g., receptor traps(such as entanercept), antibody mimetics (such as adnectins, fibronectinbased “addressable” therapeutic binding molecules from, e.g., CompoundTherapeutics, Inc.), domain antibodies (the smallest functional fragmentof a naturally occurring single-domain antibody (such as, e.g.,nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr. 15;64 (8):2853-7)).

Suitable antibody mimetics generally can be used as surrogates for theantibodies and antibody fragments described herein. Such antibodymimetics may be associated with advantageous properties (e.g., they maybe water soluble, resistant to proteolysis, and/or be nonimmunogenic).For example, peptides comprising a synthetic beta-loop structure thatmimics the second complementarity-determining region (CDR) of monoclonalantibodies have been proposed and generated. See, e.g., Saragovi et al.,Science. Aug. 16, 1991; 253 (5021):792-5. Peptide antibody mimetics alsohave been generated by use of peptide mapping to determine “active”antigen recognition residues, molecular modeling, and a moleculardynamics trajectory analysis, so as to design a peptide mimic containingantigen contact residues from multiple CDRs. See, e.g., Cassett et al.,Biochem Biophys Res Commun. Jul. 18, 2003; 307 (1):198-205. Additionaldiscussion of related principles, methods, etc., that may be applicablein the context of this invention are provided in, e.g., Fassina,Immunomethods. October 1994; 5 (2):121-9.

In various embodiments, the active pharmaceutical ingredient may haveany of a variety of activities selected from the group consisting ofanti-rheumatics, anti-neoplastic, vaccines, anti-diabetics,haematologicals, muscle relaxant, immunostimulants, anti-coagulants,bone calcium regulators, sera and gammaglobulins, anti-fibrinolytics, MStherapies, anti-anaemics, cytostatics, interferons, anti-metabolites,radiopharmaceuticals, anti-psychotics, anti-bacterials,immunosuppressants, cytotoxic antibiotics, cerebral & peripheral vasotherapeutics, nootropics, CNS drugs, dermatologicals, angiotensinantagonists, anti-spasmodics, anti-cholinergics, interferons,anti-psoriasis agents, anti-hyperlipidaemics, cardiac therapies,alkylating agents, bronchodilators, anti-coagulants,anti-inflammatories, growth hormones, and diagnostic imaging agents.

In various embodiments, the pharmaceutical composition may be selectedfrom the group consisting of VELCADE (bortezomib), STELARA(Ustekinumab), SIMPONI (golimumab), siltuximab, and AMG 403(fulranumab).

In a particular embodiment, the pharmaceutical composition comprisesVELCADE (bortezomib). The product is provided as a mannitol boronicester which, in reconstituted form, consists of the mannitol ester inequilibrium with its hydrolysis product, the monomeric boronic acid. Thedrug substance exists in its cyclic anhydride form as a trimericboroxine with the following structure.

Bortezomib is produced for intravenous injection or subcutaneous use asan antineoplastic agent for the treatment of patients with multiplemyeloma and for the treatment of patients with mantle cell lymphoma whohave received at least 1 prior therapy. The product is provided as amannitol boronic ester which, in reconstituted form, consists of themannitol ester in equilibrium with its hydrolysis product, the monomericboronic acid. The drug substance exists in its cyclic anhydride form asa trimeric boroxine.

The chemical name for bortezomib, the monomeric boronic acid, is[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronicacid and has a molecular weight of 384.24. The molecular formula isC₁₉H₂₅BN₄O₄. The solubility of bortezomib, as the monomeric boronicacid, in water is 3.3 to 3.8 mg/mL in a pH range of 2 to 6.5. Bortezomibis disclosed in U.S. Pat. Nos. 5,780,454; 6,083,903; and 6,297,217 whichare all incorporated herein by reference in their entirety.

Bortezomib is supplied as individually cartoned 10 mL vials containing3.5 mg of bortezomib as a sterile white to off-white cake or lyophilizedpowder with 35 mg mannitol, USP as an inactive ingredient.

In a particular embodiment, the pharmaceutical composition comprisesSTELARA (ustekinumab). Ustekinumab (STELARA) is a human interleukin(IL)-12 and IL-23 monoclonal antibody antagonist presently indicated forthe treatment of adult patients (18 years or older) with chronicmoderate to severe plaque psoriasis who are candidates for phototherapyor systemic therapy.

Ustekinumab, comprised of 1326 amino acids and having an estimatedmolecular mass that ranges from 148,079 to 149,690 Daltons, has thefollowing light and heavy chain variable domain sequences:

Light chain variable domain (SEQ ID NO: 1):DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIY PYTFGQGTKLEIKRHeavy chain variable domain (SEQ ID NO: 2):EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIMSPVDSDIRYSPSFQGQVTMSVDKSITTAYLQWNSLKASDTAMYYCARRRPGQGYFDFWGQGTLVTVSS

Ustekinumab is a human IgG1κ monoclonal antibody that binds with highaffinity and specificity to the p40 subunit of IL-12 and IL-23cytokines. IL-12 and IL-23 are naturally occurring cytokines that areinvolved in inflammatory and immune responses, such as natural killercell activation and CD4⁺ T-cell differentiation and activation. In invitro models, ustekinumab was shown to disrupt IL-12 and IL-23 mediatedsignaling and cytokine cascades by disrupting the interaction of thesecytokines with a shared cell-surface receptor chain, IL-12 β1. Using DNArecombinant technology, ustekinumab is produced in a well characterizedrecombinant cell line and is purified using standard bio-processingtechnology. Ustekinumab is disclosed in U.S. Pat. Nos. 6,902,734;7,166,285; 7,063,964; 7,279,157; 7,560,247; 7,887,807; 8,084,233; and8,329,171.

Ustekinumab is currently supplied as a single use prefilled syringe forsubcutaneous injection and is formulated with L-histidine andL-histidine monohydrochloride monohydrate, polysorbate 80, and sucroseto a final pH of 5.7-6.3.

In a particular embodiment, the pharmaceutical composition comprisessiltuximab (CNTO-328), a chimeric, murine-human monoclonal antibody thatbinds with high affinity and specificity to interleukin-6 (IL-6) asdescribed in U.S. Patent Publication No. 20110059080, incorporatedherein by reference. The variable region of siltuximab is derived from amurine anti-IL-6 antibody, CLB8, and the constant region is derived froma human IgG1κ molecule. Siltuzimab has the following light and heavychain variable domain sequences:

Light chain variable domain (SEQ ID NO: 3):QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSGYP YTFGGGTKLEIKHeavy chain variable domain (SEQ ID NO: 4):EVQLVESGGKLLKPGGSLKLSCAASGFTFSSFAMSWFRQSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCARGLWGYYALDYWGQGTSVTVSS

Siltuximab has been investigated for the treatment of metastatic renalcell cancer, prostate cancer, multiple myeloma, ovarian cancer, lungcancer, non-Hodgkins lymphoma, giant lymph node hyperplasia, andCastleman's disease, among other types of cancer. It is administered byintravenous infusion.

In a particular embodiment, the pharmaceutical composition comprisesSIMPONI (golimumab). Golimumab (SIMPONI, CNTO-148) is a human IgG1κmonoclonal antibody, produced by a murine hybridoma cell line withrecombinant DNA technology, that binds both the soluble andtransmembrane bioactive forms of human tumor necrosis factor (TNF) andwhich is described in U.S. Pat. Nos. 8,241,899; 7,820,169; 7,815,909;7,691,378; 7,521,206; and 7,250,165, each of which are incorporatedherein by reference. SIMPONI is provided as a 0.5 mL single-usepre-filled syringe or pre-filled pen containing 50 mg of golimumab. Thesolution is clear to slightly opalescent, colorless to light yellow.Inactive ingredients are sorbitol, histidine, histidine hydrochloridemonohydrate, polysorbate 80, and water for injections.

Golimumab forms high affinity, stable complexes with both the solubleand transmembrane bioactive forms of human tumor necrosis factor (TNF),which prevents the binding of TNF to its receptors. The binding of humanTNF by golimumab was shown to neutralize TNF-induced cell surfaceexpression of the adhesion molecules E-selectin, vascular cell adhesionmolecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1 by humanendothelial cells. TNF-induced secretion of IL-6, IL-8 andgranulocyte-macrophage colony stimulating factor (GM-CSF) by humanendothelial cells was also inhibited by golimumab.

Golimumab has the following light and heavy chain variable domainsequences:

Light Chain variable domain (SEQ ID NO: 5):Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 1               5                   10Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg         15                  20                 Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr 25                  30                  35     Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile             40                  45              Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala     50                  55                  60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr                 65                  70 Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala         75                  80                  Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro85                  90                  95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys            100                 105Heavy chain variable domain (SEQ ID NO: 6):Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val 1               5                   10Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala         15                  20 Ser Gly Phe Xaa Phe Ser Ser Tyr Ala Met His Trp 25                  30                  35Val Arg Gln Ala Pro Gly Xaa Gly Leu Glu Trp Val             40                  45Ala Xaa Xaa Xaa Xaa Asp Gly Ser Asn Lys Xaa Xaa     50                  55                  60Ala Asp Ser Val Lys Xaa Arg Phe Thr Xaa Ser Arg                 65                  70Asp Asn Xaa Lys Asn Xaa Leu Xaa Leu Gln Met Asn         75                  80Ser Leu Arg Ala Glu Asp Thr Ala Val Xaa Tyr Cys 85                  90                  95Ala Arg Asp Arg Gly Xaa Ser Ala Gly Gly Asn Tyr             100                 105Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr     110                 115                 120 Thr Val Thr Val Ser Ser                125

Xaa at position 28 is Ile or Thr. Xaa at position 43 is Lys or Asn. Xaaat position 50 is Ile, Phe or Val. Xaa at position 51 is Ile or Met. Xaaat position 52 is Ser or Leu. Xaa at position 53 is Tyr or Phe. Xaa atposition 59 is Lys or Tyr. Xaa at position 60 is Ser or Tyr. Xaa atposition 66 is Asp or Gly. Xaa at position 70 is Val or Ile. Xaa atposition 75 is Ser or Pro. Xaa at position 78 is Thr or Ala. Xaa atposition 80 is Tyr or Phe. Xaa at position 94 is Tyr or Phe. Xaa atposition 102 is Ile or Val.

Golimumab is approved for the treatment of rheumatoid arthritis,psoriatic arthritis, and ankylosing spondylitis in adult patients.

In a particular embodiment, the pharmaceutical composition comprisesfulranumab. Fulranumab (AMG403; immunoglobulin G2, anti-(human nervegrowth factor) (human monoclonal 4D4 heavy chain), disulfide with humanmonoclonal 4D4 light chain, dimer; or immunoglobulin G2, anti-(humanbeta-nerve growth factor (beta-NGF)); human monoclonal 4D4 γ2 heavychain (137-214′)-disulfide with human monoclonal 4D4 κ light chain,dimer (225-225″:226-226″:229-229″:232-232″)-tetrakisdisulfide) is amonoclonal antibody directed against nerve growth factor (NGF) withpotential analgesic activity. Fulranumab has a molecular weight of145.39 kDa. Fulranumab is disclosed in U.S. Pat. Nos. 7,601,818;8,106,167; and 8,198,410, each of which are incorporated herein byreference in their entirety.

The light and heavy chain sequences of fulranumab are as follows:

Light chain (SEQ ID NO: 7):AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain (SEQ ID NO: 8):EVQLVESGGGLVQPGGSLRLSCAASGFTLRSYSMNWVRQAPGKGLEWVSYISRSSHTIFYADSVKGRFTISRDNAKNSLYLQMDSLRDEDTAMYYCARVYSSGWHVSDYFDYWGQGILVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Upon administration, fulranumab binds to NGF, preventing its binding toand activation of the NGF receptors TrkA and p75NTR Inhibition of theNGF pathway may prevent the perception of pain and may induce analgesia.NGF, a neurotrophic factor that plays a key role in neuropathic andinflammatory-induced pain, promotes hyperalgesia and allodynia.

Degradation and Stability of Pharmaceutical Compositions

According to the present invention, delamination resistantpharmaceutical containers comprising a glass composition provide forimproved resistance to degradation of, improved stability of, improvedresistance to inactivation of, and improved maintenance of levels of apharmaceutical composition having at least one active pharmaceuticalingredient, for example, VELCADE (bortezomib), STELARA (ustekinumab),SIMPONI (golimumab), siltuximab, and AMG 403 (fulranumab).

In one embodiment of the present invention, the delamination resistantpharmaceutical containers provide improved stability to pharmaceuticalcompositions contained therein, for example, VELCADE (bortezomib),STELARA (ustekinumab), SIMPONI (golimumab), siltuximab, and AMG 403(fulranumab). As used herein, the term “stability” refers to the abilityof an active pharmaceutical ingredient to essentially retain itsphysical, chemical and conformational identity and integrity uponstorage in the pharmaceutical containers of the invention. Stability isassociated with the ability of an active pharmaceutical ingredient toretain its potency and efficacy over a period of time. Instability of anactive pharmaceutical ingredient may be associated with, for example,chemical or physical degradation, fragmentation, conformational change,increased toxicity, aggregation (e.g., to form higher order polymers),deglycosylation, modification of glycosylation, oxidation, hydrolysis,or any other structural, chemical or physical modification. Suchphysical, chemical and/or conformational changes often result in reducedactivity or inactivation of the active pharmaceutical ingredient, forexample, such that at least one biological activity of the activepharmaceutical ingredient is reduced or eliminated. Alternatively or inaddition, such physical, chemical and/or conformational changes oftenresult in the formation of structures toxic to the subject to whom thepharmaceutical composition is administered.

The pharmaceutical containers of the present invention maintainstability of the pharmaceutical compositions, in part, by minimizing oreliminating delamination of the glass composition which forms, at leastin part, the pharmaceutical container. In addition, the pharmaceuticalcontainers of the present invention maintain stability of thepharmaceutical compositions, in part, by reducing or preventing theinteraction of the active pharmaceutical ingredient with thepharmaceutical container and/or delaminated particles resultingtherefrom. By minimizing or eliminating delamination and, further, byreducing or preventing interaction, the pharmaceutical containersthereby reduce or prevent the destabilization of the activepharmaceutical ingredient as found in, for example, VELCADE(bortezomib), STELARA (ustekinumab), SIMPONI (golimumab), siltuximab,and AMG 403 (fulranumab).

The pharmaceutical containers of the present invention provide theadditional advantage of preventing loss of active pharmaceuticalingredients. For example, by reducing or preventing the interaction ofand, thus, the adherence of, the active pharmaceutical ingredient withthe pharmaceutical container and/or delaminated particles resultingtherefrom, the level of active pharmaceutical ingredient available foradministration to a subject is maintained, as found in, for example,VELCADE (bortezomib), STELARA (ustekinumab), SIMPONI (golimumab),siltuximab, and AMG 403 (fulranumab). In one embodiment of the presentinvention, the pharmaceutical composition has a high pH. According tothe present invention, it has been discovered that high pHs serve toincrease delamination of glass compositions. Accordingly, thepharmaceutical containers of the present invention are particularlysuitable for storing and maintaining pharmaceutical compositions havinga high pH, for example, pharmaceutical compositions having a pH betweenabout 7 and about 11, between about 7 and about 10, between about 7 andabout 9, or between about 7 and about 8.

In additional embodiments, the pharmaceutical containers of the presentinvention are particularly suitable for storing and maintainingpharmaceutical compositions having phosphate or citrate based buffers.According to the present invention, it has been discovered thatphosphate or citrate based buffers serve to increase delamination ofglass compositions. According in particular embodiments, thepharmaceutical composition includes a buffer comprising a salt ofcitrate, e.g., sodium citrate, or SSC. In other embodiments, thepharmaceutical composition includes a buffer comprising a salt ofphosphate, e.g., mono or disodium phosphate.

In additional embodiments, the pharmaceutical containers of the presentinvention are particularly suitable for storing and maintaining activepharmaceutical ingredient that needs to be subsequently formulated. Inother embodiments, the pharmaceutical containers of the presentinvention are particularly suitable for storing and maintaining alyophilized pharmaceutical composition or active pharmaceuticalingredient that requires reconstitution, for example, by addition ofsaline.

Assaying for Delamination of Pharmaceutical Containers

As noted above, delamination may result in the release of silica-richglass flakes into a solution contained within the glass container afterextended exposure to the solution. Accordingly, the resistance todelamination may be characterized by the number of glass particulatespresent in a solution contained within the glass container afterexposure to the solution under specific conditions. In order to assessthe long-term resistance of the glass container to delamination, anaccelerated delamination test was utilized. The test consisted ofwashing the glass container at room temperature for 1 minute anddepyrogenating the container at about 320° C. for 1 hour. Thereafter asolution of 20 mM glycine with a pH of 10 in water is placed in theglass container to 80-90% fill, the glass container is closed, andrapidly heated to 100° C. and then heated from 100° C. to 121° C. at aramp rate of 1 deg/min at a pressure of 2 atmospheres. The glasscontainer and solution are held at this temperature for 60 minutes,cooled to room temperature at a rate of 0.5 deg./min and the heatingcycle and hold are repeated. The glass container is then heated to 50°C. and held for two days for elevated temperature conditioning. Afterheating, the glass container is dropped from a distance of at least 18″onto a firm surface, such as a laminated tile floor, to dislodge anyflakes or particles that are weakly adhered to the inner surface of theglass container.

Thereafter, the solution contained in the glass container is analyzed todetermine the number of glass particles present per liter of solution.Specifically, the solution from the glass container is directly pouredonto the center of a Millipore Isopore Membrane filter (Millipore#ATTP02500 held in an assembly with parts #AP1002500 and #M000025A0)attached to vacuum suction to draw the solution through the filterwithin 10-15 seconds. Particulate flakes are then counted bydifferential interference contrast microscopy (DIC) in the reflectionmode as described in “Differential interference contrast (DIC)microscopy and modulation contrast microscopy” from Fundamentals oflight microscopy and digital imaging. New York: Wiley-Liss, pp 153-168.The field of view is set to approximately 1.5 mm×1.5 mm and particleslarger than 50 microns are counted manually. There are 9 suchmeasurements made in the center of each filter membrane in a 3×3 patternwith no overlap between images. A minimum of 100 mL of solution istested. As such, the solution from a plurality of small containers maybe pooled to bring the total amount of solution to 100 mL. If thecontainers contain more than 10 mL of solution, the entire amount ofsolution from the container is examined for the presence of particles.For containers having a volume greater than 10 mL, the test is repeatedfor a trial of 10 containers formed from the same glass compositionunder the same processing conditions and the result of the particlecount is averaged for the 10 containers to determine an average particlecount. Alternatively, in the case of small containers, the test isrepeated for a trial of 10 sets of 10 mL of solution, each of which isanalyzed and the particle count averaged over the 10 sets to determinean average particle count. Averaging the particle count over multiplecontainers accounts for potential variations in the delaminationbehavior of individual containers. Table 7 summarizes some non-limitingexamples of sample volumes and numbers of containers for testing isshown below:

TABLE 7 Table of Exemplary Test Specimens Nominal Minimum Total VialVial Max Solution Number of solution Capacity Volume per Vial Vials in aNumber of Tested (mL) (mL) (mL) Trial Trials (mL) 2 4 3.2 4 10 128 3.5 75.6 2 10 112 4 6 4.8 3 10 144 5 10 8 2 10 160 6 10 8 2 10 160 8 11.5 9.22 10 184 10 13.5 10.8 1 10 108 20 26 20.8 1 10 208 30 37.5 30 1 10 30050 63 50.4 1 10 504

It should be understood that the aforementioned test is used to identifyparticles which are shed from the interior wall(s) of the glasscontainer due to delamination and not tramp particles present in thecontainer from forming processes or particles which precipitate from thesolution enclosed in the glass container as a result of reactionsbetween the solution and the glass. Specifically, delamination particlesmay be differentiated from tramp glass particles based on the aspectratio of the particle (i.e., the ratio of the width of the particle tothe thickness of the particle). Delamination produces particulate flakesor lamellae which are irregularly shaped and are typically >50 μm indiameter but often >200 μm. The thickness of the flakes is usuallygreater than about 100 nm and may be as large as about 1 μm. Thus, theminimum aspect ratio of the flakes is typically >50. The aspect ratiomay be greater than 100 and sometimes greater than 1000. Particlesresulting from delamination processes generally have an aspect ratiowhich is generally greater than about 50. In contrast, tramp glassparticles will generally have a low aspect ratio which is less thanabout 3. Accordingly, particles resulting from delamination may bedifferentiated from tramp particles based on aspect ratio duringobservation with the microscope. Validation results can be accomplishedby evaluating the heel region of the tested containers. Uponobservation, evidence of skin corrosion/pitting/flake removal, asdescribed in “Nondestructive Detection of Glass Vial Inner SurfaceMorphology with Differential Interference Contrast Microscopy” fromJournal of Pharmaceutical Sciences 101(4), 2012, pages 1378-1384, isnoted.

In the embodiments described herein, glass containers which average lessthan 3 glass particles with a minimum width of 50 μm and an aspect ratioof greater than 50 per trial following accelerated delamination testingare considered “delamination resistant.” In the embodiments describedherein, glass containers which average less than 2 glass particles witha minimum width of 50 μm and an aspect ratio of greater than 50 pertrial following accelerated delamination testing are considered“delamination-stable.” In the embodiments described herein, glasscontainers which average less than 1 glass particle with a minimum widthof 50 μm and an aspect ratio of greater than 50 per trial followingaccelerated delamination testing are considered “delamination-proof.” Inthe embodiments described herein, glass containers which have 0 glassparticles with a minimum width of 50 μm and an aspect ratio of greaterthan 50 per trial following accelerated delamination testing areconsidered “delamination-free”.

Assessing Stability of Pharmaceutical Compositions

As set forth above, any of a variety of active pharmaceuticalingredients can be incorporated within the claimed pharmaceuticalcontainer including, for example, a small molecule, a polypeptidemimetic, a biologic, an antisense RNA, a small interfering RNA (siRNA),etc. These active ingredients degrade in varying manners and, thus,assessing the stability thereof in the pharmaceutical containers of thepresent invention requires different techniques.

Depending on the nature of the active pharmaceutical ingredient, thestability, maintenance and/or continued efficacy of the pharmaceuticalcompositions contained within the delamination resistant pharmaceuticalcontainers of the present invention can be evaluated as follows.

Biologics API are often susceptible to degradation and/or inactivationarising from various factors, including pH, temperature, temperaturecycling, light, humidity, etc. Biologics API are further susceptible todegradation, inactivation or loss, arising from interaction of thepharmaceutical composition with the pharmaceutical container, ordelaminants leeching therefrom. For example, biologics may undergophysical degradation which may render the resulting pharmaceuticalcomposition inactive, toxic or insufficient to achieve the desiredeffect. Alternatively, or in addition, biologics may undergo structuralor conformational changes that can alter the activity of the API, withor without degradation. For example, proteins may undergo unfoldingwhich can result in effective loss and inactivity of the API.Alternatively, or in addition, biologics may adhere to the surface ofthe container, thereby rendering the API administered to the subjectinsufficient to achieve the desired effect, e.g., therapeutic effect.

(i) General Methods for Investigation of Biologic Compound Degradation

Depending on the size and complexity of the biologic, methods foranalysis of degradation of non-biologic, small molecule API may beapplied to biologics. For example, peptides and nucleic acids can beanalyzed using any of a number of chromatography and spectrometrytechniques applicable to small molecules to determine the size of themolecules, either with or without protease or nuclease digestion.However, as proper secondary and tertiary structures are required forthe activity of biologics, particularly protein biologics, confirmationof molecular weight is insufficient to confirm activity of biologics.Protein biologics containing complex post-translational modifications,e.g., glycosylation, are less amenable to analysis using chromatographyand spectrometry. Moreover, complex biologics, e.g., vaccines which caninclude complex peptide mixtures, attenuated or killed viruses, orkilled cells, are not amenable to analysis by most chromatography orspectrometry methods.

(ii) In Vitro Functional Assays for Investigation of Compound Stability

One or more in vitro assays, optionally in combination with one or morein vivo assays, can be used to assess the stability and activity of theAPI. Functional assays to determine API stability can be selected basedon the structural class of the API and the function of the API.Exemplary assays are provided below to confirm the activity of the APIafter stability and/or stress testing. It is understood that assaysshould be performed with the appropriate controls (e.g., vehiclecontrols, control API not subject to stress or stability testing) with asufficient number of dilutions and replicate samples to provide datawith sufficient statistical significance to detect changes in activityof 10% or less, preferably 5% or less, 4% or less, more preferably 3% orless, 2% or less, or 1% or less, as desired. Such considerations in theart are well understood.

For example, antibody based therapeutics, regardless of the disease orcondition to be treated, can be assayed for stability and activity usingassays that require specific binding of the antibody to its cognateantigen, e.g., ELISA. The antigen used in the ELISA should have theappropriate conformational structure as would be found in vivo. Antibodybased API are used, for example, for the treatment of cancer andinflammatory diseases including autoimmune diseases.

ELISA assays to assay the concentration of a protein biologic API arecommercially available from a number of sources, e.g., R&D Systems, BDBiosciences, AbCam, Pierce, Invitrogen.

API are frequently targeted to receptors, particularly cell surfacereceptors. Receptor binding assays can be used to assess the activity ofsuch agents. API that bind cell surface receptors can be agonists,antagonists or allosteric modulators. API that bind cell surfacereceptors need not bind the same location as the native ligand tomodulate, for example, inhibit or enhance, signaling through thereceptor. Depending on the activity of the API, an appropriate assay canbe selected, e.g., assay for stimulation of receptor signaling when theAPI is a receptor agonist; and inhibition assay in which binding of anagonist, e g, inhibition of activation by a receptor agonist by the API.Such assays can be used regardless of the disease(s) or condition(s) tobe treated with the API. Modulation of cellular activity, e.g., cellproliferation, apoptosis, cell migration, modulation of expression ofgenes or proteins, differentiation, tube formation, etc. is assayedusing routine methods. In other assay methods, a reporter construct isused to indicate activation of the receptor. Such methods are routine inthe art. APIs that bind to cell surface receptors are used, for example,as anti-cancer agents, anti-diabetic agents, anti-inflammatory agentsfor the treatment of inflammatory mediated diseases including autoimmunedisorders, anti-angiogenic agents, anti-cholinergic agents, bone calciumregulators, muscle and vascular tension regulators, and psychoactiveagents.

Modulators of cell proliferation can be assayed for activity using acell proliferation assays. For example, cell proliferation is inducedusing anti-anemic agents or stimulators of hematopoietic cell growth.Anti-proliferative agents, e.g., cytotoxic agents, anti-neoplasticagents, chemotherapeutic agents, cytostatic agents, antibiotic agents,are used to inhibit growth of various cell types. Some anti-inflammatoryagents also act by inhibiting proliferation of immune cells, e.g., blastcells. In proliferation assays, replicate wells containing the samenumber of cells are cultured in the presence of the API. The effect ofthe API is assessed using, for example, microscopy or fluorescenceactivated cell sorting (FACS) to determine if the number of cells in thesample increased or decreased in response to the presence of the API. Itis understood that the cell type selected for the proliferation assay isdependent on the specific API to be tested.

Modulators of angiogenesis can be assayed using cell migration and/ortube formation assays. For cell migration assays, human vascularendothelial cells (HUVECs) are cultured in the presence of the API intranswell devices. Migration of cells through the device at the desiredtime intervals is assessed. Alternatively, 3-dimensional HUVECs culturesin MATRIGEL can be assessed for tube formation. Anti-angiogenic agentsare used, for example, for the treatment of cancer, maculardegeneration, and diabetic retinopathy.

Anti-inflammatory API can be assayed for their effects on immune cellstimulation as determined, for example, by modulation of one or more ofcytokine expression and secretion, antigen presentation, migration inresponse to cytokine or chemokine stimulation, and immune cellproliferation. In such assays, immune cells are cultured in the presenceof the API and changes in immune cell activity are determined usingroutine methods in the art, e.g., ELISA and cell imaging and counting.

Anti-diabetic API can be assayed for their effects on insulin signaling,including insulin signaling in response to modulated glucose levels, andinsulin secretion. Insulin signaling can be assessed by assessing kinaseactivation in response to exposure to insulin and/or modulation ofglucose levels. Insulin secretion can be assessed by ELISA assay.

Modulators of blood clotting, i.e., fibrinolytics, anti-fibrinolytics,and anti-coagulants, can be assayed for their effects using an INR assayon serum by measuring prothrombin time to determine a prothrombin ratio.Time to formation of a clot is assayed in the presence or absence of theAPI.

Modulators of muscle or vascular tone can be assayed for their effectsusing vascular or muscle explants. The tissue can be placed in a caliperfor detection of changes in length and/or tension. Whole coronaryexplants can be used to assess the activity of API on heart. The tissueis contacted with the API, and optionally agents to alter vascular tone(e.g., K⁺, Ca⁺⁺). The effects of the API on length and/or tension of thevasculature or muscle is assessed.

Psychoactive agents can act by modulation of neurotransmitter releaseand/or recycling. Neuronal cells can be incubated in the presence of anAPI and stimulated to release neurotransmitters. Neurotransmitter levelscan be assessed in the culture medium collected at defined time pointsto detect alterations in the level of neurotransmitter present in themedia. Neurotransmitters can be detected, for example, using ELISA,LC/MS/MS, or by preloading the vesicles with radioactiveneurotransmitters to facilitate detection.

(iii) In Vivo Assays for Investigation of Compound Stability

In addition to in vitro testing for compound stability, API can also betested in vivo to confirm the stability of the API after storage and/orstress testing. For example, some API are not amenable to testing usingin vitro assays due to the complexity of the disease state or thecomplexity of the response required. For example, psychoactive agents,e.g., antipsychotic agents, anti-depressant agents, nootropic agents,immunosuppressant agents, vasotherapeutic agents, muscular dystrophyagents, central nervous system modulating agents, antispasmodic agents,bone calcium regenerating agents, anti-rheumatic agents,anti-hyperlipidemic agents, hematopoietic proliferation agents, growthfactors, vaccine agents, and imaging agents, may not be amenable to fullfunctional characterization using in vitro models. Moreover, for someagents, factors that may not alter in vitro activity may alter activityin vivo, e.g., antibody variable domains may be sufficient to blocksignaling through a receptor, but the Fc domains may be required forefficacy in the treatment of disease. Further, changes in stability mayresult in changes in pharmacokinetic properties of an API (e.g.,half-life, serum protein binding, tissue distribution, CNSpermeability). Finally, changes in stability may result in thegeneration of toxic degradation or reaction products that would not bedetected in vivo. Therefore, confirmation of pharmacokinetic andpharmacodynamic properties and toxicity in vivo is useful in conjunctionwith stability and stress testing.

(iv) Pharmacokinetic Assays

Pharmacokinetics includes the study of the mechanisms of absorption anddistribution of an administered drug, the rate at which a drug actionbegins and the duration of the effect, the chemical changes of thesubstance in the body (e.g. by metabolic enzymes such as CYP or UGTenzymes) and the effects and routes of excretion of the metabolites ofthe drug. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as the ADME scheme:

-   -   Absorption—the process of a substance entering the blood        circulation.    -   Distribution—the dispersion or dissemination of substances        throughout the fluids and tissues of the body.    -   Metabolism (or Biotransformation)—the irreversible        transformation of parent compounds into daughter metabolites.    -   Excretion—the removal of the substances from the body. In rare        cases, some drugs irreversibly accumulate in body tissue.    -   Elimination is the result of metabolism and excretion.

Pharmacokinetics describes how the body affects a specific drug afteradministration. Pharmacokinetic properties of drugs may be affected byelements such as the site of administration and the dose of administereddrug, which may affect the absorption rate. Such factors cannot be fullyassessed using in vitro models.

The specific pharmacokinetic properties to be assessed for a specificAPI in stability testing will depend, for example, on the specific APIto be tested. In vitro pharmacokinetic assays can include assays of drugmetabolism by isolated enzymes or by cells in culture. However,pharmacokinetic analysis typically requires analysis in vivo.

As pharmacokinetics are not concerned with the activity of the drug, butinstead with the absorption, distribution, metabolism, and excretion ofthe drug, assays can be performed in normal subjects, rather thansubjects suffering from a disease or condition for which the API istypically administered, by administration of a single dose of the API tothe subject. However, if the subject to be treated with the API has acondition that would alter the metabolism or excretion of the API, e.g.,liver disease, kidney disease, testing of the API in an appropriatedisease model may be useful. Depending on the half life of the compound,samples (e.g., serum, urine, stool) are collected at predetermined timepoints for at least two, preferably three half-lives of the API, andanalyzed for the presence of the API and metabolic products of the API.At the end of the study, organs are harvested and analyzed for thepresence of the API and metabolic products of the API. Thepharmacokinetic properties of the API subjected to stability and/orstress testing are compared to API not subjected to stability or stresstesting and other appropriate controls (e.g., vehicle control). Changesin pharmacokinetic properties as a result of stability and/or stresstesting are determined.

(v) Pharmacodynamic Assays

Pharmacodynamics includes the study of the biochemical and physiologicaleffects of drugs on the body or on microorganisms or parasites within oron the body and the mechanisms of drug action and the relationshipbetween drug concentration and effect. Due to the complex nature of manydisease states and the actions of many API, the API should be tested invivo to confirm the desired activity of the agent. Mouse models for alarge variety of disease states are known and commercially available(see, e.g.,jaxmice.jax.org/query/f?p=205:1:989373419139701::::P1_ADV:1). A numberof induced models of disease are also known. Agents can be tested on theappropriate animal model to demonstrate stability and efficacy of theAPI on modulating the disease state.

(vi) Specific Immune Response Assay

Vaccines produce complex immune responses that are best assessed invivo. The specific potency assay for a vaccine depends, at least inpart, on the specific vaccine type. The most accurate predictions arebased on mathematical modeling of biologically relevantstability-indicating parameters. For complex vaccines, e.g., whole cellvaccines, whole virus vaccines, complex mixtures of antigens,characterization of each component biochemically may be difficult, ifnot impossible. For example, when using a live, attenuated virusvaccine, the number of plaque forming units (e.g., mumps, measles,rubella, smallpox) or colony forming units (e.g., S. typhi, TY21a) aredetermined to confirm potency after storage. Chemical and physicalcharacterization (e.g., polysaccharide and polysaccharide-proteinconjugate vaccines) is performed to confirm the stability and activityof the vaccine. Serological response in animals to inactivated toxinsand/or animal protection against challenge (e.g., rabies, anthrax,diphtheria, tetanus) is performed to confirm activity of vaccines of anytype, particularly when the activity of the antigen has beeninactivated. In vivo testing of vaccines subjected to stability and/orstress testing is performed by administering the vaccine to a subjectusing the appropriate immunization protocol for the vaccine, anddetermining the immune response by detection of specific immune cellsthat respond to stimulation with the antigen or pathogen, detection ofantibodies against the antigen or pathogen, or protection in an immunechallenge. Such methods are well known in the art. Vaccines include, butare not limited to, meningococcal B vaccine, hepatitis A and B vaccines,human papillomavirus vaccine, influenza vaccine, herpes zoster vaccine,and pneumococcal vaccine.

(vii) Toxicity Assays

Degradation of API can result in in the formation of toxic agents.Toxicity assays include the administration of doses, typically farhigher than would be used for therapeutic applications, to detect thepresence of toxic products in the API. Toxicity assays can be performedin vitro and in vivo and are frequently single, high dose experiments.After administration of the compound, in addition to viability, organsare harvested and analyzed for any indication of toxicity, especiallyorgans involved with clearance of API, e.g., liver, kidneys, and thosefor which damage could be catastrophic, e.g., heart, brain. Thetoxicologic properties of the API subjected to stability and/or stresstesting are compared to API not subjected to stability or stress testingand other appropriate controls (e.g., vehicle control). Changes intoxicologic properties as a result of stability and/or stress testingare determined.

In accordance with present invention, the degradation, alteration ordepletion of API contained within a delamination resistantpharmaceutical container of the present invention can be assessed by avariety of physical techniques. Indeed, in various aspects of theinvention, the stability and degradation caused by the interaction ofAPI with the container or delaminants thereof, or changes inconcentration or amount of the API in a container can be assessed usingtechniques as follows. Such methods include, e.g., X-Ray Diffraction(XRPD), Thermal Analysis (such as Differential Scanning calorimetry(DSC), Thermogravimetry (TG) and Hot-Stage Microscopy (HSM),chromatography methods (such as High-Performance Liquid Chromatography(HPLC), Column Chromatography (CC), Gas Chromatography (GC), Thin-LayerChromatography (TLC), and Super Critical Phase Chromatograph (SFC)),Mass Spectroscopy (MS), Capillary Electrophoresis (CE), AtomicSpectroscopy (AS), vibrational spectroscopy (such as InfraredSpectroscopy (IR)), Luminescence Spectroscopy (LS), and Nuclear MagneticResonance Spectroscopy (NMR).

In the case of pharmaceutical formulations where the API is not insolution or needs to be reconstituted into a different medium, XRPD maybe a method for analyzing degradation. In ideal cases, every possiblecrystalline orientation is represented equally in a non-liquid sample.

Powder diffraction data is usually presented as a diffractogram in whichthe diffracted intensity I is shown as function either of the scatteringangle 2θ or as a function of the scattering vector q. The lattervariable has the advantage that the diffractogram no longer depends onthe value of the wavelength λ. Relative to other methods of analysis,powder diffraction allows for rapid, non-destructive analysis ofmulti-component mixtures without the need for extensive samplepreparation. Deteriorations of an API may be analyzed using this method,e.g., by comparing the diffraction pattern of the API to a knownstandard of the API prior to packaging.

Thermal methods of analysis may include, e.g., differential scanningcalorimetry (DSC), thermogravimetry (TG), and hot-stage microscopy(HSM). All three methods provide information upon heating the sample.Depending on the information required, heating can be static or dynamicin nature.

Differential scanning calorimetry monitors the energy required tomaintain the sample and a reference at the same temperature as they areheated. A plot of heat flow (W/g or J/g) versus temperature is obtained.The area under a DSC peak is directly proportional to the heat absorbedor released and integration of the peak results in the heat oftransition.

Thermogravimetry (TG) measures the weight change of a sample as afunction of temperature. A total volatile content of the sample isobtained, but no information on the identity of the evolved gas isprovided. The evolved gas must be identified by other methods, such asgas chromatography, Karl Fisher titration (specifically to measurewater), TG—mass spectroscopy, or TG—infrared spectroscopy. Thetemperature of the volatilization and the presence of steps in the TGcurve can provide information on how tightly water or solvent is held inthe lattice. If the temperature of the TG volatilization is similar toan endothermic peak in the DSC, the DSC peak is likely due or partiallydue to volatilization. It may be necessary to utilize multipletechniques to determine if more than one thermal event is responsiblefor a given DSC peak.

Hot-Stage Microscopy (HSM) is a technique that supplements DSC and TG.Events observed by DSC and/or TG can be readily characterized by HSM.Melting, gas evolution, and solid-solid transformations can bevisualized, providing the most straightforward means of identifyingthermal events. Thermal analysis can be used to determine the meltingpoints, recrystallizations, solid-state transformations, decompositions,and volatile contents of pharmaceutical materials.

Other methods to analyze degradation or alteration of API and excipientsare infrared (IR) and Raman spectroscopy. These techniques are sensitiveto the structure, conformation, and environment of organic compounds.Infrared spectroscopy is based on the conversion of IR radiation intomolecular vibrations. For a vibration to be IR-active, it must involve achanging molecular dipole (asymmetric mode). For example, vibration of adipolar carbonyl group is detectable by IR spectroscopy. Whereas IR hasbeen traditionally used as an aid in structure elucidation, vibrationalchanges also serve as probes of intermolecular interactions in solidmaterials.

Raman spectroscopy is based on the inelastic scattering of laserradiation with loss of vibrational energy by a sample. A vibrationalmode is Raman active when there is a change in the polarizability duringthe vibration. Symmetric modes tend to be Raman-active. For example,vibrations about bonds between the same atom, such as in alkynes, can beobserved by Raman spectroscopy.

NMR spectroscopy probes atomic environments based on the differentresonance frequencies exhibited by nuclei in a strong magnetic field.Many different nuclei are observable by the NMR technique, but those ofhydrogen and carbon atoms are most frequently studied. Solid-state NMRmeasurements are extremely useful for characterizing the crystal formsof pharmaceutical solids. Nuclei that are typically analyzed with thistechnique include those of 13C, 31P, 15N, 25Mg, and 23Na.

Chromatography is a general term applied to a wide variety of separationtechniques based on the sample partitioning between a moving phase,which can be a gas, liquid, or supercritical fluid, and a stationaryphase, which may be either a liquid or a solid. Generally, the crux ofchromatography lies in the highly selective chemical interactions thatoccur in both the mobile and stationary phases. For example, dependingon the API and the separation required, one or more of absorption,ion-exchange, size-exclusion, bonded phase, reverse, or normal phasestationary phases may be employed.

Mass spectrometry (MS) is an analytical technique that works by ionizingchemical compounds to generate charged molecules or molecule fragmentsand measuring their mass-to-charge ratios. Based on this analysismethod, one can determine, e.g., the isotopic composition of elements inan API and determine the structure of the API by observing itsfragmentation pattern.

It would be understood that the foregoing methods do not represent acomprehensive list of means by which one can analyze possibledeteriorations, alterations, or concentrations of certain APIs.Therefore, it would be understood that other methods for determining thephysical amounts and/or characteristics of an API may be employed.Additional methods may include, but are not limited to, e.g., CapillaryElectrophoresis (CE), Atomic Spectroscopy (AS), and LuminescenceSpectroscopy (LS).

EXAMPLES

The embodiments of the delamination resistant pharmaceutical containersdescribed herein will be further clarified by the following examples.

Example 1

Six exemplary inventive glass compositions (compositions A-F) wereprepared. The specific compositions of each exemplary glass compositionare reported below in Table 8. Multiple samples of each exemplary glasscomposition were produced. One set of samples of each composition wasion exchanged in a molten salt bath of 100% KNO₃ at a temperature of450° C. for at least 5 hours to induce a compressive layer in thesurface of the sample. The compressive layer had a surface compressivestress of at least 500 MPa and a depth of layer of at least 45 μm.

The chemical durability of each exemplary glass composition was thendetermined utilizing the DIN 12116 standard, the ISO 695 standard, andthe ISO 720 standard described above. Specifically, non-ion exchangedtest samples of each exemplary glass composition were subjected totesting according to one of the DIN 12116 standard, the ISO 695standard, or the ISO 720 standard to determine the acid resistance, thebase resistance or the hydrolytic resistance of the test sample,respectively. The hydrolytic resistance of the ion exchanged samples ofeach exemplary composition was determined according to the ISO 720standard. The average results of all samples tested are reported belowin Table 8.

As shown in Table 8, exemplary glass compositions A-F all demonstrated aglass mass loss of less than 5 mg/dm² and greater than 1 mg/dm²following testing according to the DIN 12116 standard with exemplaryglass composition E having the lowest glass mass loss at 1.2 mg/dm².Accordingly, each of the exemplary glass compositions were classified inat least class S3 of the DIN 12116 standard, with exemplary glasscomposition E classified in class S2. Based on these test results, it isbelieved that the acid resistance of the glass samples improves withincreased SiO₂ content.

Further, exemplary glass compositions A-F all demonstrated a glass massloss of less than 80 mg/dm² following testing according to the ISO 695standard with exemplary glass composition A having the lowest glass massloss at 60 mg/dm². Accordingly, each of the exemplary glass compositionswere classified in at least class A2 of the ISO 695 standard, withexemplary glass compositions A, B, D and F classified in class A1. Ingeneral, compositions with higher silica content exhibited lower baseresistance and compositions with higher alkali/alkaline earth contentexhibited greater base resistance.

Table 8 also shows that the non-ion exchanged test samples of exemplaryglass compositions A-F all demonstrated a hydrolytic resistance of atleast Type HGA2 following testing according to the ISO 720 standard withexemplary glass compositions C-F having a hydrolytic resistance of TypeHGA1. The hydrolytic resistance of exemplary glass compositions C-F isbelieved to be due to higher amounts of SiO₂ and the lower amounts ofNa₂O in the glass compositions relative to exemplary glass compositionsA and B.

Moreover, the ion exchanged test samples of exemplary glass compositionsB-F demonstrated lower amounts of extracted Na₂O per gram of glass thanthe non-ion exchanged test samples of the same exemplary glasscompositions following testing according to the ISO 720 standard.

TABLE 8 Composition and Properties of Exemplary Glass CompositionsComposition in mole % A B C D E F SiO₂ 70.8 72.8 74.8 76.8 76.8 77.4Al₂O₃ 7.5 7 6.5 6 6 7 Na₂O 13.7 12.7 11.7 10.7 11.6 10 K₂O 1 1 1 1 0.10.1 MgO 6.3 5.8 5.3 4.8 4.8 4.8 CaO 0.5 0.5 0.5 0.5 0.5 0.5 SnO₂ 0.2 0.20.2 0.2 0.2 0.2 DIN 12116 3.2 2.0 1.7 1.6 1.2 1.7 (mg/dm²) S3 S3 S3 S3S2 S3 classification ISO 695 60.7 65.4 77.9 71.5 76.5 62.4 (mg/dm²) A1A1 A2 A1 A2 A1 classification ISO 720 100.7 87.0 54.8 57.5 50.7 37.7 (ugNa₂O/ HGA2 HGA2 HGA1 HGA1 HGA1 HGA1 g glass) classification ISO 720 60.351.9 39.0 30.1 32.9 23.3 (with IX) HGA1 HGA1 HGA1 HGA1 HGA1 HGA1 (ugNa₂O/ g glass) classification

Example 2

Three exemplary inventive glass compositions (compositions G-I) andthree comparative glass compositions (compositions 1-3) were prepared.The ratio of alkali oxides to alumina (i.e., Y:X) was varied in each ofthe compositions in order to assess the effect of this ratio on variousproperties of the resultant glass melt and glass. The specificcompositions of each of the exemplary inventive glass compositions andthe comparative glass compositions are reported in Table 9. The strainpoint, anneal point, and softening point of melts formed from each ofthe glass compositions were determined and are reported in Table 2. Inaddition, the coefficient of thermal expansion (CTE), density, andstress optic coefficient (SOC) of the resultant glasses were alsodetermined and are reported in Table 9. The hydrolytic resistance ofglass samples formed from each exemplary inventive glass composition andeach comparative glass composition was determined according to the ISO720 Standard both before ion exchange and after ion exchange in a moltensalt bath of 100% KNO₃ at 450° C. for 5 hours. For those samples thatwere ion exchanged, the compressive stress was determined with afundamental stress meter (FSM) instrument, with the compressive stressvalue based on the measured stress optical coefficient (SOC). The FSMinstrument couples light into and out of the birefringent glass surface.The measured birefringence is then related to stress through a materialconstant, the stress-optic or photoelastic coefficient (SOC or PEC) andtwo parameters are obtained: the maximum surface compressive stress (CS)and the exchanged depth of layer (DOL). The diffusivity of the alkaliions in the glass and the change in stress per square root of time werealso determined.

TABLE 9 Glass properties as a function of alkali to alumina ratioComposition Mole % G H I 1 2 3 SiO₂ 76.965 76.852 76.962 76.919 76.96077.156 Al₂O₃ 5.943 6.974 7.958 8.950 4.977 3.997 Na₂O 11.427 10.4739.451 8.468 12.393 13.277 K₂O 0.101 0.100 0.102 0.105 0.100 0.100 MgO4.842 4.878 4.802 4.836 4.852 4.757 CaO 0.474 0.478 0.481 0.480 0.4680.462 SnO₂ 0.198 0.195 0.197 0.197 0.196 0.196 Strain (° C.) 578 616 654683 548 518 Anneal (° C.) 633 674 716 745 600 567 Softening (° C.) 892946 1003 1042 846 798 Expansion 67.3 64.3 59.3 55.1 71.8 74.6 (10⁻⁷ K⁻¹)Density (g/cm³) 2.388 2.384 2.381 2.382 2.392 2.396 SOC (nm/ 3.127 3.1813.195 3.232 3.066 3.038 mm/Mpa) ISO720 88.4 60.9 47.3 38.4 117.1 208.1(non-IX) ISO720 25.3 26 20.5 17.8 57.5 102.5 (IX450° C.-5 hr) R₂O/Al₂O₃1.940 1.516 1.200 0.958 2.510 3.347 CS@t = 0 708 743 738 655 623 502(MPa) CS/{square root over (t)} (MPa/ −35 −24 −14 −7 −44 −37 hr^(1/2)) D(μm²/hr) 52.0 53.2 50.3 45.1 51.1 52.4

The data in Table 9 indicates that the alkali to alumina ratio Y:Xinfluences the melting behavior, hydrolytic resistance, and thecompressive stress obtainable through ion exchange strengthening. Inparticular, FIG. 1 graphically depicts the strain point, anneal point,and softening point as a function of Y:X ratio for the glasscompositions of Table 9. FIG. 1 demonstrates that, as the ratio of Y:Xdecreases below 0.9, the strain point, anneal point, and softening pointof the glass rapidly increase. Accordingly, to obtain a glass which isreadily meltable and formable, the ratio Y:X should be greater than orequal to 0.9 or even greater than or equal to 1.

Further, the data in Table 2 indicates that the diffusivity of the glasscompositions generally decreases with the ratio of Y:X. Accordingly, toachieve glasses can be rapidly ion exchanged in order to reduce processtimes (and costs) the ratio of Y:X should be greater than or equal to0.9 or even greater than or equal to 1.

Moreover, FIG. 2 indicates that for a given ion exchange time and ionexchange temperature, the maximum compressive stresses are obtained whenthe ratio of Y:X is greater than or equal to about 0.9, or even greaterthan or equal to about 1, and less than or equal to about 2,specifically greater than or equal to about 1.3 and less than or equalto about 2.0. Accordingly, the maximum improvement in the load bearingstrength of the glass can be obtained when the ratio of Y:X is greaterthan about 1 and less than or equal to about 2. It is generallyunderstood that the maximum stress achievable by ion exchange will decaywith increasing ion-exchange duration as indicated by the stress changerate (i.e., the measured compressive stress divided by the square rootof the ion exchange time). FIG. 2 generally shows that the stress changerate decreases as the ratio Y:X decreases.

FIG. 3 graphically depicts the hydrolytic resistance (y-axis) as afunction of the ratio Y:X (x-axis). As shown in FIG. 3, the hydrolyticresistance of the glasses generally improves as the ratio Y:X decreases.

Based on the foregoing it should be understood that glasses with goodmelt behavior, superior ion exchange performance, and superiorhydrolytic resistance can be achieved by maintaining the ratio Y:X inthe glass from greater than or equal to about 0.9, or even greater thanor equal to about 1, and less than or equal to about 2.

Example 3

Three exemplary inventive glass compositions (compositions J-L) andthree comparative glass compositions (compositions 4-6) were prepared.The concentration of MgO and CaO in the glass compositions was varied toproduce both MgO-rich compositions (i.e., compositions J-L and 4) andCaO-rich compositions (i.e., compositions 5-6). The relative amounts ofMgO and CaO were also varied such that the glass compositions haddifferent values for the ratio (CaO/(CaO+MgO)). The specificcompositions of each of the exemplary inventive glass compositions andthe comparative glass compositions are reported below in Table 10. Theproperties of each composition were determined as described above withrespect to Example 2.

TABLE 10 Glass properties as function of CaO content Composition Mole %J K L 4 5 6 SiO₂ 76.99 77.10 77.10 77.01 76.97 77.12 Al₂O₃ 5.98 5.975.96 5.96 5.97 5.98 Na₂O 11.38 11.33 11.37 11.38 11.40 11.34 K₂O 0.100.10 0.10 0.10 0.10 0.10 MgO 5.23 4.79 3.78 2.83 1.84 0.09 CaO 0.07 0.451.45 2.46 3.47 5.12 SnO₂ 0.20 0.19 0.19 0.19 0.19 0.19 Strain (° C.) 585579 568 562 566 561 Anneal (° C.) 641 634 620 612 611 610 Softening (°C.) 902 895 872 859 847 834 Expansion 67.9 67.1 68.1 68.8 69.4 70.1(10⁻⁷ K⁻¹) Density (g/cm³) 2.384 2.387 2.394 2.402 2.41 2.42 SOCnm/mm/Mpa 3.12 3.08 3.04 3.06 3.04 3.01 ISO720 (non-IX) 83.2 83.9 86 8688.7 96.9 ISO720 (IX450° 29.1 28.4 33.2 37.3 40.1 C.-5 hr) Fraction ofRO as 0.014 0.086 0.277 0.465 0.654 0.982 CaO CS@t = 0 (MPa) 707 717 713689 693 676 CS/{square root over (t)} (MPa/hr^(1/2)) −36 −37 −39 −38 −43−44 D (μm²/hr) 57.2 50.8 40.2 31.4 26.4 20.7

FIG. 4 graphically depicts the diffusivity D of the compositions listedin Table 10 as a function of the ratio (CaO/(CaO+MgO)). Specifically,FIG. 4 indicates that as the ratio (CaO/(CaO+MgO)) increases, thediffusivity of alkali ions in the resultant glass decreases therebydiminishing the ion exchange performance of the glass. This trend issupported by the data in Table 10 and FIG. 5. FIG. 5 graphically depictsthe maximum compressive stress and stress change rate (y-axes) as afunction of the ratio (CaO/(CaO+MgO)). FIG. 5 indicates that as theratio (CaO/(CaO+MgO)) increases, the maximum obtainable compressivestress decreases for a given ion exchange temperature and ion exchangetime. FIG. 5 also indicates that as the ratio (CaO/(CaO+MgO)) increases,the stress change rate increases (i.e., becomes more negative and lessdesirable).

Accordingly, based on the data in Table 10 and FIGS. 4 and 5, it shouldbe understood that glasses with higher diffusivities can be produced byminimizing the ratio (CaO/(CaO+MgO)). It has been determined thatglasses with suitable diffusivities can be produced when the(CaO/(CaO+MgO)) ratio is less than about 0.5. The diffusivity values ofthe glass when the (CaO/(CaO+MgO)) ratio is less than about 0.5decreases the ion exchange process times needed to achieve a givencompressive stress and depth of layer. Alternatively, glasses withhigher diffusivities due to the ratio (CaO/(CaO+MgO)) may be used toachieve a higher compressive stress and depth of layer for a given ionexchange temperature and ion exchange time.

Moreover, the data in Table 10 also indicates that decreasing the ratio(CaO/(CaO+MgO)) by increasing the MgO concentration generally improvesthe resistance of the glass to hydrolytic degradation as measured by theISO 720 standard.

Example 4

Three exemplary inventive glass compositions (compositions M-O) andthree comparative glass compositions (compositions 7-9) were prepared.The concentration of B₂O₃ in the glass compositions was varied from 0mol. % to about 4.6 mol. % such that the resultant glasses had differentvalues for the ratio B₂O₃/(R₂O—Al₂O₃). The specific compositions of eachof the exemplary inventive glass compositions and the comparative glasscompositions are reported below in Table 11. The properties of eachglass composition were determined as described above with respect toExamples 2 and 3.

TABLE 11 Glass properties as a function of B₂O₃ content Composition Mole% M N O 7 8 9 SiO₂ 76.860 76.778 76.396 74.780 73.843 72.782 Al₂O₃ 5.9645.948 5.919 5.793 5.720 5.867 B₂O₃ 0.000 0.214 0.777 2.840 4.443 4.636Na₂O 11.486 11.408 11.294 11.036 10.580 11.099 K₂O 0.101 0.100 0.1000.098 0.088 0.098 MgO 4.849 4.827 4.801 4.754 4.645 4.817 CaO 0.4920.480 0.475 0.463 0.453 0.465 SnO₂ 0.197 0.192 0.192 0.188 0.183 0.189Strain (° C.) 579 575 572 560 552 548 Anneal (° C.) 632 626 622 606 597590 Softening (° C.) 889 880 873 836 816 801 Expansion 68.3 67.4 67.465.8 64.1 67.3 (10⁻⁷ K⁻¹) Density (g/cm³) 2.388 2.389 2.390 2.394 2.3922.403 SOC (nm/ 3.13 3.12 3.13 3.17 3.21 3.18 mm/MPa) ISO720 86.3 78.868.5 64.4 52.7 54.1 (non-IX) ISO720 (IX450° 32.2 30.1 26 24.7 22.6 26.7C.-5 hr) B₂O₃/ 0.000 0.038 0.142 0.532 0.898 0.870 (R₂O—Al₂O₃) CS@t = 0(MPa) 703 714 722 701 686 734 CS/{square root over (t )} (MPa/ −38 −38−38 −33 −32 −39 hr^(1/2)) D (μm²/hr) 51.7 43.8 38.6 22.9 16.6 15.6

FIG. 6 graphically depicts the diffusivity D (y-axis) of the glasscompositions in Table 11 as a function of the ratio B₂O₃/(R₂O—Al₂O₃)(x-axis) for the glass compositions of Table 11. As shown in FIG. 6, thediffusivity of alkali ions in the glass generally decreases as the ratioB₂O₃/(R₂O—Al₂O₃) increases.

FIG. 7 graphically depicts the hydrolytic resistance according to theISO 720 standard (y-axis) as a function of the ratio B₂O₃/(R₂O—Al₂O₃)(x-axis) for the glass compositions of Table 11. As shown in FIG. 6, thehydrolytic resistance of the glass compositions generally improves asthe ratio B₂O₃/(R₂O—Al₂O₃) increases.

Based on FIGS. 6 and 7, it should be understood that minimizing theratio B₂O₃/(R₂O—Al₂O₃) improves the diffusivity of alkali ions in theglass thereby improving the ion exchange characteristics of the glass.Further, increasing the ratio B₂O₃/(R₂O—Al₂O₃) also generally improvesthe resistance of the glass to hydrolytic degradation. In addition, ithas been found that the resistance of the glass to degradation in acidicsolutions (as measured by the DIN 12116 standard) generally improveswith decreasing concentrations of B₂O₃. Accordingly, it has beendetermined that maintaining the ratio B₂O₃/(R₂O—Al₂O₃) to less than orequal to about 0.3 provides the glass with improved hydrolytic and acidresistances as well as providing for improved ion exchangecharacteristics.

It should now be understood that the glass compositions described hereinexhibit chemical durability as well as mechanical durability followingion exchange. These properties make the glass compositions well suitedfor use in various applications including, without limitation,pharmaceutical packaging materials.

Example 5 Determining the Presence and Amount of Glass Flakes inPharmaceutical Solutions

The resistance to delamination may be characterized by the number ofglass particulates present in a pharmaceutical solution contained withina glass container described herein after. In order to assess thelong-term resistance of the glass container to delamination, anaccelerated delamination test is utilized. The test consists of washingthe glass container at room temperature for 1 minute and depyrogenatingthe container at about 320° C. for 1 hour. Thereafter a pharmaceuticalsolution is placed in the glass container to 80-90% full, the glasscontainer is closed, and rapidly heated to, for example, 100° C. andthen heated from 100° C. to 121° C. at a ramp rate of 1 deg/min at apressure of 2 atmospheres. The glass container and solution are held atthis temperature for 60 minutes, cooled to room temperature at a rate of0.5 deg./min and the heating cycle and hold are repeated. The glasscontainer is then heated to 50° C. and held for two days for elevatedtemperature conditioning. After heating, the glass container is droppedfrom a distance of at least 18″ onto a firm surface, such as a laminatedtile floor, to dislodge any flakes or particles that are weakly adheredto the inner surface of the glass container.

Thereafter, the pharmaceutical solution contained in the glass containeris analyzed to determine the number of glass particles present per literof solution. Specifically, the solution from the glass container isdirectly poured onto the center of a Millipore Isopore Membrane filter(Millipore #ATTP02500 held in an assembly with parts #AP1002500 and#M000025A0) attached to vacuum suction to draw the solution through thefilter within 10-15 seconds. Particulate flakes are then counted bydifferential interference contrast microscopy (DIC) in the reflectionmode as described in “Differential interference contrast (DIC)microscopy and modulation contrast microscopy” from Fundamentals oflight microscopy and digital imaging. New York: Wiley-Liss, pp 153-168.The field of view is set to approximately 1.5 mm×1.5 mm and particleslarger than 50 microns are counted manually. There are 9 suchmeasurements made in the center of each filter membrane in a 3×3 patternwith no overlap between images. A minimum of 100 mL of solution istested. As such, the solution from a plurality of small containers maybe pooled to bring the total amount of solution to 100 mL. If thecontainers contain more than 10 mL of solution, the entire amount ofsolution from the container is examined for the presence of particles.For containers having a volume greater than 10 mL containers, the testis repeated for a trial of 10 containers formed from the same glasscomposition under the same processing conditions and the result of theparticle count is averaged for the 10 containers to determine an averageparticle count. Alternatively, in the case of small containers, the testis repeated for a trial of 10 sets of 10 mL of solution, each of whichis analyzed and the particle count averaged over the 10 sets todetermine an average particle count. Averaging the particle count overmultiple containers accounts for potential variations in thedelamination behavior of individual containers.

It should be understood that the aforementioned test is used to identifyparticles which are shed from the interior wall(s) of the glasscontainer due to delamination and not tramp particles present in thecontainer from forming processes. Specifically, delamination particleswill be differentiated from tramp glass particles based on the aspectratio of the particle (i.e., the ratio of the width of the particle tothe thickness of the particle). Delamination produces particulate flakesor lamellae which are irregularly shaped and are typically >50 μm indiameter but often >200 μm. The thickness of the flakes is usuallygreater than about 100 nm and may be as large as about 1 μm. Thus, theminimum aspect ratio of the flakes is typically >50. The aspect ratiomay be greater than 100 and sometimes greater than 1000. Particlesresulting from delamination processes generally have an aspect ratiowhich is generally greater than about 50. In contrast, tramp glassparticles will generally have a low aspect ratio which is less thanabout 3. Accordingly, particles resulting from delamination may bedifferentiated from tramp particles based on aspect ratio duringobservation with the microscope. Validation results can be accomplishedby evaluating the heel region of the tested containers. Uponobservation, evidence of skin corrosion/pitting/flake removal, asdescribed in “Nondestructive Detection of Glass Vial Inner SurfaceMorphology with Differential Interference Contrast Microscopy” fromJournal of Pharmaceutical Sciences 101(4), 2012, pages 1378-1384, isnoted.

Using this method, pharmaceutical compositions can be tested for thepresence of glass flakes and various compositions can be compared toeach other to assess the safety of various pharmaceutical compositions.

Example 6 Stability Testing of Pharmaceutical Compositions

Stability studies are part of the testing required by the FDA and otherregulatory agencies. Stability studies should include testing of thoseattributes of the API that are susceptible to change during storage andare likely to influence quality, safety, and/or efficacy. The testingshould cover, as appropriate, the physical, chemical, biological, andmicrobiological attributes of the API (e.g., small molecule or biologictherapeutic agent) in the container with the closure to be used forstorage of the agent. If the API is formulated as a liquid by themanufacturer, the final formulation should be assayed for stability. Ifthe API is formulated as an agent for reconstitution by the end userusing a solution provided by the manufacturer, both the API and thesolution for reconstitution are preferably tested for stability as theseparate packaged components (e.g., the API subjected to storagereconstituted with solution for reconstitution not subject to storage,API not subject to storage reconstituted with a solution subject tostorage, and both API and solution subject to storage). This isparticularly the case when the solution for reconstitution includes anactive agent (e.g., an adjuvant for reconstitution of a vaccine).

In general, a substance API should be evaluated under storage conditions(with appropriate tolerances) that test its thermal stability and, ifapplicable, its sensitivity to moisture. The storage conditions and thelengths of studies chosen should be sufficient to cover storage,shipment, and subsequent use.

API should be stored in the container(s) in which the API will beprovided to the end user (e.g., vials, ampules, syringes, injectabledevices). Stability testing methods provided herein refer to samplesbeing removed from the storage or stress conditions indicated. Removalof a sample preferably refers to removing an entire container from thestorage or stress conditions. Removal of a sample should not beunderstood as withdrawing a portion of the API from the container asremoval of a portion of the API from the container would result inchanges of fill volume, gas environment, etc. At the time of testing theAPI subject to stability and/or stress testing, portions of the samplessubject to stability and/or stress testing can be used for individualassays.

The long-term testing should cover a minimum of 12 months' duration onat least three primary batches at the time of submission and should becontinued for a period of time sufficient to cover the proposed retestperiod. Additional data accumulated during the assessment period of theregistration application should be submitted to the authorities ifrequested. Data from the accelerated storage condition and, ifappropriate, from the intermediate storage condition can be used toevaluate the effect of short-term excursions outside the label storageconditions (such as might occur during shipping).

Long-term, accelerated, and, where appropriate, intermediate storageconditions for API are detailed in the sections below. The general caseshould apply if the API is not specifically covered by a subsequentsection. It is understood that the time points for analysis indicated inthe table are suggested end points for analysis. Interim analysis can bepreformed at shorter time points (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 months). For API to be labeled as stable for storage for more than12 months, time points beyond 12 months can be assessed (e.g., 15, 18,21, 24 months). Alternative storage conditions can be used if justified.

TABLE 12 General Conditions for Stability Analysis Time points StudyStorage condition for analysis Long-term Long-term* 12 months  25° C. ±2° C./60% RH ± 5% RH or 30° C. ± 2° C./65% RH ± 5% RH Intermediate 30°C. ± 2° C./65% RH ± 5% RH 6 months Accelerated 40° C. ± 2° C./75% RH ±5% RH 6 months

TABLE 13 Conditions for Stability Analysis for Storage in a RefrigeratorMinimum time period covered by Study Storage condition data atsubmission Long-term 5° C. ± 3° C. 12 months  Accelerated 25° C. ± 2°C./60% RH ± 5% RH 6 months

TABLE 14 Conditions for Stability Analysis for Storage in a FreezerMinimum time period covered by Study Storage condition data atsubmission Long-term −20° C. ± 5° C. 12 months

Storage condition for API intended to be stored in a freezer, testing ona single batch at an elevated temperature (e.g., 5° C.±3° C. or 25°C.±2° C.) for an appropriate time period should be conducted to addressthe effect of short-term excursions outside the proposed label storagecondition (e.g., stress during shipping or handling, e.g., increasedtemperature, multiple freeze-thaw cycles, storage in a non-uprightorientation, shaking, etc.).

The assays performed to assess stability of an API include assays tothat are used across most APIs to assess the physical properties of theAPI, e.g., degradation, pH, color, particulate formation, concentration,toxicity, etc. Assays to detect the general properties of the API arealso selected based on the chemical class of the agent, e.g.,denaturation and aggregation of protein based API. Assays to detect thepotency of the API, i.e., the ability of the API to achieve its intendedeffect as demonstrated by the quantitative measurement of an attributeindicative of the clinical effect as compared to an appropriate control,are selected based on the activity of the particular agent. For example,the biological activity of the API, e.g., enzyme inhibitor activity,cell killing activity, anti-inflammatory activity, coagulationmodulating activity, etc., is measured using in vitro and/or in vivoassays such as those provided herein. Pharmacokinetic and toxicologicalproperties of the API are also assessed using methods known in the art,such as those provided herein.

Example 7 Analysis of Adherence to Glass Vials

Changes in the surface of glass can result in changes in the adherenceof API to glass. The amount of agent in samples withdrawn from glassvials are tested at intervals to determine if the concentration of theAPI in solution changes over time. API are incubated in containers asdescribed in the stability testing and/or stress testing methodsprovided in Example 6. Preferably, the API is incubated both in standardglass vials with appropriate closures and glass vials such as thoseprovided herein. At the desired intervals, samples are removed andassayed to determine the concentration of the API in solution. Theconcentration of the API is determined using methods and controlsappropriate to the API. The concentration of the API is preferablydetermined in conjunction with at least one assay to confirm that theAPI, rather than degradation products of the API, is detected. In thecase of biologics in which the conformational structure of the biologicagent is essential to its function of the API, the assays forconcentration of the biologic are preferably preformed in conjunctionwith an assay to confirm the structure of the biologic (e.g., activityassay).

For example, in the cases of small molecule APIs, the amount of agentpresent is determined, for example, by mass spectrometry, optionally incombination with liquid chromatography, as appropriate, to separate theagent from any degradation products that may be present in the sample.

For protein based biologic APIs, the concentration of the API isdetermined, for example, using ELISA assay. Chromatography methods areused in conjunction with methods to determine protein concentration toconfirm that protein fragments or aggregates are not being detected bythe ELISA assay.

For nucleic acid biologic APIs, the concentration of the API isdetermined, for example, using quantitative PCR when the nucleic acidsare of sufficient length to permit detection by such methods.Chromatography methods are used to determine both the concentration andsize of nucleic acid based API.

For viral vaccine APIs, the concentration of the virus is determined,for example, using colony formation assays.

Example 8 Analysis of Pharmacokinetic Properties

Pharmacokinetics is concerned with the analysis of absorption,distribution, metabolism, and excretion of API. Storage and stress canpotentially affect the pharmacokinetic properties of various API. Toassess pharmacokinetics of API subject to stability and/or stresstesting, agents are incubated in containers as described in Example 6.Preferably, the API are incubated both in standard glass vials withappropriate closures and glass vials such as those provided herein. Atthe desired intervals, samples are removed and assayed.

The API is delivered to subjects by the typical route of delivery forthe API (e.g., injection, oral, topical). As pharmacokinetics areconcerned with the absorption and elimination of the API, normalsubjects are typically used to assess pharmacokinetic properties of theAPI. However, if the API is to be used in subjects with compromisedability to absorb or eliminate the API (e.g., subjects with liver orkidney disease), testing in an appropriate disease model may beadvantageous. Depending on the half life of the compound, samples (e.g.,blood, urine, stool) are collected at predetermined time points (e.g., 0min, 30 min, 60 min, 90 min, 120 min, 4 hours, 6 hours, 12 hours, 24hours, 36 hours, 48 hours, etc.) for at least two, preferably threehalf-lives of the API, and analyzed for the presence of the API andmetabolic products of the API. At the end of the study, organs areharvested and analyzed for the presence of the API and metabolicproducts of the API.

The results are analyzed using an appropriate model selected based on,at least, the route of administration of the API. The pharmacokineticproperties of the API subjected to stability and/or stress testing arecompared to API not subjected to stability or stress testing and otherappropriate controls (e.g., vehicle control). Changes, if any, inpharmacokinetic properties as a result of storage of the API under eachcondition are determined.

Example 9 Analysis of Toxicity Profiles

Storage of API can result in alterations of toxicity of API as a resultof reactivity of the API with the container, leeching of agents from thecontainer, delamination resulting in particulates in the agent, reactionof the API molecules with each other or components of the storagebuffer, or other causes.

Agents are incubated in containers as described in the stability testingand/or stress testing methods provided in Example 6. Preferably, the APIis incubated both in standard glass vials with appropriate closures andglass vials such as those provided herein. At the desired intervals,samples are removed and assayed to determine the toxicity the API. Thetoxicity of the API is determined using methods and controls appropriateto the API. In vitro and in vivo testing can be used alone or incombination to assess changes in toxicity of agents as a result ofstorage or stress.

In in vitro assays, cell lines are grown in culture and contacted withincreasing concentrations of API subjected to stability and/or stresstesting for predetermined amounts of time (e.g., 12, 24, 36, 48, and 72hours). Cell viability is assessed using any of a number of routine orcommercially available assays. Cells are observed, for example, bymicroscopy or using fluorescence activated cell sorting (FACS) analysisusing commercially available reagents and kits. For example,membrane-permeant calcein AM is cleaved by esterases in live cells toyield cytoplasmic green fluorescence, and membrane-impermeant ethidiumhomodimer-1 labels nucleic acids of membrane-compromised cells with redfluorescence. Membrane-permeant SYTO 10 dye labels the nucleic acids oflive cells with green fluorescence, and membrane-impermeant DEAD Red dyelabels nucleic acids of membrane-compromised cells with redfluorescence. A change in the level of cell viability is detectedbetween the cells contacted with API subjected to stress and/orstability testing in standard glass vials as compared to the glass vialsprovided herein and appropriate controls (e.g., API not subject tostability testing, vehicle control).

In vivo toxicity assays are performed in animals. Typically preliminaryassays are performed on normal subjects. However, if the disease orcondition to be treated could alter the susceptibility of the subject totoxic agents (e.g., decreased liver function, decreased kidneyfunction), toxicity testing in an appropriate model of the disease orcondition can be advantageous. One or more doses of agents subjected tostability and/or stress testing are administered to animals. Typically,doses are far higher (e.g., 5 times, 10 times) the dose that would beused therapeutically and are selected, at least in part, on the toxicityof the API not subject to stability and/or stress testing. However, forthe purpose of assaying stability of API, the agent can be administeredat a single dose that is close to (e.g., 70%-90%), but not at, a dosethat would be toxic for the API not subject to stability or stresstesting. In single dose studies, after administration of the API subjectto stress and/or stability testing (e.g., 12 hours, 24 hours, 48 hours,72 hours), during which time blood, urine, and stool samples may becollected. In long term studies, animals are administered a lower dose,closer to the dose used for therapeutic treatment, and are observed forchanges indicating toxicity, e.g., weight loss, loss of appetite,physical changes, or death. In both short and long term studies, organsare harvested and analyzed to determine if the API is toxic. Organs ofmost interest are those involved in clearance of the API, e.g., liverand kidneys, and those for which toxicity would be most catastrophic,e.g., heart, brain. An analysis is performed to detect a change intoxicity between the API subjected to stress and/or stability testing instandard glass vials as compared to the glass vials provided herein, ascompared to API not subject to stability and/or stress testing andvehicle control. Changes, if any, in toxicity properties as a result ofstorage of the API under each condition are determined.

Example 10 Analysis of Pharmacodynamic Profiles

Pharmacodynamics includes the study of the biochemical and physiologicaleffects of drugs on the body or on microorganisms or parasites within oron the body and the mechanisms of drug action and the relationshipbetween drug concentration and effect. Mouse models for a large varietyof disease states are known and commercially available (see, e.g.,jaxmice.jax.org/query/f?p=205:1:989373419139701::::P1_ADV:1). A numberof induced models of disease are also known.

Agents are incubated in containers as described in the stability testingand/or stress testing methods provided in Example 6. Preferably, thesamples are incubated both in standard glass vials with appropriateclosures and glass vials such as those provided herein. At the desiredintervals, samples are removed and assayed for pharmacodynamic activityusing known animal models. Exemplary mouse models for testing thevarious classes of agents indicated are known in the art.

The mouse is treated with the API subject to stability and/or stresstesting. The efficacy of the API subject to stability and/or stresstesting to treat the appropriate disease or condition is assayed ascompared to API not subject to stability and/or stress testing andvehicle control. Changes, if any, in pharmacodynamic properties as aresult of storage of the API under each condition are determined.

Example 11 Confirmation of Stability and Integrity of Bortezomib

Storage and stress can potentially affect the biological activity ofsmall molecule API. Assays to test the activity of 26S proteasomeinhibitors, such as bortezomib, are known in the art. To assesspharmacokinetics of API subject to stability and/or stress testing,agents are incubated in containers as described in Example 6.Preferably, the API are incubated both in standard glass vials withappropriate closures and glass vials such as those provided herein. Atthe desired intervals, samples are removed and assayed using knownmethods such as antigen presentation assays, protein degradation assays,corticosterone-induced cachexia assays, and cell adhesion moleculeexpression assays provided herein.

Antigen Presentation Assay in LB27.4 Cells

Antigen presentation is dependent upon 26S proteasome activity. Antigenpresentation is inhibited in a dose dependent manner by bortezomib. Anexemplary antigen presentation assay for 26S proteasome activity isprovided in Rock et al., 1994 (Inhibitors of the proteasome block thedegradation of most cell proteins and the generation of peptidespresented on MHC class I molecules. Cell 78:761-771, incorporated hereinby reference) and is provided herein.

Briefly, LB27.4 cells are incubated for 1 hr at 37° C. in Optimem mediawith various concentrations of bortezomib subject or not subject tostability and/or stress testing as in Example 6. Antigens ovalbumin (30mg/ml) or SIINFEKL peptide (SEQ ID NO: 9) are introduced into the cellsby electroporation. The ovalbumin must be processed on the proteasome tobe presented on the cell surface, whereas the SIINFEKL peptide (SEQ IDNO: 9) can be loaded into an MHC I complex without processing to act asa positive control for antigen presentation.

After introduction of the peptides into the cells by electroporation,the cells are either fixed immediately with paraformaldehyde (1%) for 10min at 25° C. or are incubated for 1-2 hr at 37° C. in Optimem in thecontinued presence or absence of inhibitors and then fixed withparaformaldehyde. The presence of peptide-MHC complexes on the surfaceof LB27.4 cells is assayed by measuring the amount of interleukin-2(IL-2) produced by the ovalbumin-K^(b)-specific T-T hybridoma RF33.70after stimulation with LB27.4 cells in duplicate cultures using routinemethods (see, e.g., Rock and Benacerraf, 1983 Inhibition ofantigen-specific T lymphocyte activation by structurally related lr genecontrolled polymers: evidence of specific competition for accessory cellantigen-presentation. J. Exp. Med. 157, 1618-1634.). Changes in theamount of IL-2 production between the bortezomib subject or not subjectto stability and/or stress testing are indicative of changes in theactivity of bortezomib in response to stability and/or stress testing.

Protein Degradation Assay in C2C12 Cells

C2C12 cells (a mouse myoblast line) are labelled for 48 hrs with³⁵S-methionine. The cells are then washed and preincubated for 2 hrs inthe same media supplemented with 2 mM unlabelled methionine. The mediais removed and replaced with a fresh aliquot of the preincubation mediacontaining 50% serum, and various concentrations of bortezomib subjector not subject to stability and/or stress testing as in Example 6. Themedia is then removed and made up to 10% TCA and centrifuged. The TCAsoluble radioactivity is counted Inhibition of proteolysis is calculatedas the percent decrease in TCA soluble radioactivity. The amount ofprotein degradation observed in samples in which the bortezomib wassubject or was not subject to stability and/or stress testing arecompared to detect changes in activity.

Corticosterone-Induced Cachexia Assay in Rats

Rats are stabilized on a diet free from 3-methylhistidine and thenplaced in metabolic cages for collection of 24-hour urine samples. Aftertwo days of urine collections to determine basal 3-methylhistidineoutput, the rats are treated with daily subcutaneous injections ofcorticosterone (100 mg/kg). Starting on the second day of corticosteronetreatment, some of the rats are also treated with various concentrationsof bortezomib subject or not subject to stability and/or stress testingas in Example 6, administered via a subcutaneous osmotic pump at a doserate of approximately 120 μg/kg body weight/day. Control rats receivevehicle only (25% DMSO/75% PEG (200)), administered in a similarfashion. Urinary output of 3-methylhistidine induced in response tocorticosterone treatment is monitored to determine differences, if any,between the samples of bortezomib subject or not subject to stabilityand/or stress testing.

Cell Adhesion Molecule Expression Assay in HUVE Cells

HUVECs in microtiter plates are exposed to various concentrations ofbortezomib subject or not subject to stability and/or stress testing asin Example 6, for 1 hour, prior to the addition of 100 U/mL TNF-α. Cellsurface binding assays are performed at 4° C. using saturatingconcentrations of monoclonal antibodies specific for the cell adhesionmolecules and fluorescently-conjugated secondary antibodies. Fluorescentimmunoassays for E-selectin and I-CAM are performed at 4 hours, thosefor V-CAM at 16 hours. Cell-surface expression I-CAM, V-CAM, andE-selectin on TNF-α stimulated HUVECs are quantitated to determinedifferences, if any, between cells contacted with samples of bortezomibsubject, or not subject to, stability and/or stress testing.

Example 12 Confirmation of Stability and Integrity of Antibody API bySize Determination (Ustekinumab (STELARA®); Siltuximab; Golimumab(SIMPONI); and Fulranumab

Antibody based API are used in the treatment of a number of diseases andconditions. The activity of antibody based API is dependent upon theability of the antibody to specifically bind to its target antigen and,depending on the specific agent and condition to be treated, for theantibody-antigen complex to be recognized and cleared by the body. Theseactivities are dependent upon proper subunit structure of the antibodywhich can be altered during storage or stress of the antibody. Antibodyactivity can be assessed, for example, using methods general to proteinAPI and antibodies, such as column chromatography and gelelectrophoresis and staining, or using methods specific to the antigenbound by the antibody as provided below. Column chromatography can alsobe used to assess protein concentration and degradation.

For example, ustekinumab is a human IgG1κ monoclonal antibody that bindswith high affinity and specificity the p40 subunit of the IL-12 andIL-23 cytokines, and is presently indicated for the treatment of chronicmoderate to severe plaque psoriasis.

Siltuximab is a chimeric, murine-human monoclonal antibody that bindswith high affinity and specificity to interleukin-6 (IL-6) that has beeninvestigated for the treatment of a variety of cancers includingmetastatic renal cell cancer, prostate cancer, multiple myeloma, ovariancancer, lung cancer, non-Hodgkins lymphoma, giant lymph nodehyperplasia, and Castleman's disease.

Golimumab is a human IgG1κ monoclonal antibody that binds to both thesoluble and transmembrane bioactive forms of human tumor necrosis factor(TNF) that is used for the treatment of arthritis and active ankylosingspondylitis.

Fulranumab is a human monoclonal antibody that binds to human nervegrowth factor (NGF) with potential analgesic activity.

Antibody based API are incubated in containers as described in thestability testing and/or stress testing methods provided in Example 6.Preferably, the samples are incubated both in standard glass vials withappropriate closures and glass vials such as those provided herein. Atthe desired intervals, samples are removed and assayed to determineantibody based API size and subunit structure, for example, bychromatography and/or gel electrophoresis methods.

Chromatography Methods for Size Detection

Samples of antibody based API subject or not subject to stability and/orstress testing are diluted and subject to analysis by size exclusionchromatography (SEC), preferably SEC-high performance liquidchromatography (SEC-HPLC). Chromatography columns are available from anumber of commercial vendors (e.g., WATERS, BIO-RAD). Size exclusionchromatography columns separate compounds by gel filtrationchromatography. The working range of the gel is the volume between thevoid volume, those solutes which elute first, and the totally includedvolume, those solutes which elute last. Protein aggregates; complexes,e.g., dimers; monomers; and fragments, can be detected by photometry forabsorbance at 350-500 nm or by UV detection at 280 nm.

The specific column type, column size, solvent type, flow rate, anddetector type can be selected based on the specific API to be analyzed.Different solvents can be selected to detect monomer or higher orderstructures. Naturally occurring IgG antibodies and antibodies produce inhybridoma cells are composed of two light chains and two heavy chainsheld together by disulfide bonds. Column chromatography methods can beused to assess the antibody structure as a tetramer, under non-reducingconditions, or as the monomer subunits, under reducing conditions.Therapeutic antibodies include other antibody formats such as singlechain antibodies in which the light and heavy chain are expressed as asingle peptide. Antibody-antigen binding can also be analyzed byincubating an excess of antigen with the antibody prior to columnchromatography. The amount of antibody-antigen complex is determined bya shift in molecular weight of the antibody, and the ratio of antibodythat does or does not bind antigen is determined An excess of unboundantigen is observed.

The relative amounts of aggregates, tetramers, monomers, degradationproducts, and antigen binding are determined in an antibody API samplethat has undergone stability and/or stress testing and compared to thecontrol antibody API sample that has not undergone stability and/orstress testing. The total protein concentration is also determined Achange in the amount of aggregate, tetramer, monomer, or degradationproducts; a change in antigen binding; and/or a change in total proteinconcentration, indicate a change in the antibody as a result of stressand/or stability testing.

Gel Electrophoresis Methods for Size Detection

Samples of antibody based API subject or not subject to stability and/orstress testing are diluted and resolved by gel electrophoresis. SDS-PAGEis used to determine protein size and aggregate formation. Non-reducinggel electrophoresis is used to analyze antibody tetramer structure.Antibodies resolved by gel electrophoresis are detected using sensitivestaining methods, such as silver stain. Gel electrophoresis and silverstaining methods are known in the art. A change in the amount ofaggregate, tetramer, monomer, or degradation products; and/or a changein total protein concentration, indicate a change in the antibody as aresult of stress and/or stability testing.

Example 13 Confirmation of Stability and Integrity of Protein API byAntibody Binding

A number of antibody-based API are known including, but not limited to,ustekinumab, siltuximab, golimumab, and fulranumab. Antibodyconcentration and conformation can be assessed using assays based onantibody binding, such as an enzyme-linked immunosorbent assay (ELISA).

Antibody based API are incubated in containers as described in thestability testing and/or stress testing methods provided in Example 6.Preferably, the samples are incubated both in standard glass vials withappropriate closures and glass vials such as those provided herein. Atthe desired intervals, samples are removed and assayed using ELISA. Asandwich ELISA method, described briefly herein, can be used to probethe structure of the antigen biding domain as well as the immunoglobulindomain. Variations based on the specific antibody API and ELISA formatused, as well as selection of appropriate dilutions and controls, arewell within the ability of those of skill in the art.

For the sake of clarity, the description of sandwich ELISA methods belowfor the detection and characterization of an antibody based API refersto the TNF-binding antibody golimumab, its antigen TNF, and anti-IgG1κsecondary antibody. However, it is understood that any of antibody API,cognate antigen, and appropriate secondary antibody can be used in themethods.

A first antibody capable of binding TNF simultaneously with golimumab isbound to a solid support, e.g., the well of a 96- or 384-well plate.Tumor necrosis factor is then added to the well. Serial dilutions ofsamples of golimumab, subject or not subject to stability and/or stresstesting, are diluted and contacted with the complex attached to thesolid support and unbound antibody is removed by washing. Therefore, theamount of golimumab present in the well is dependent upon the amount ofgolimumab in the sample that was subject to or not subject to stabilityand/or stress testing with a correctly folded antigen binding domain tobind TNF. A detectably labeled secondary antibody (e.g., horse radishperoxidase, alkaline phosphatase labeled antibody) that binds to humanIgG1κ is added to the well. The amount of secondary antibody in thesample is detected using an appropriate method based on the detectablelabel. The amount of golimumab detected in the samples subject to or notsubject to stability and/or stress testing are compared.

Example 14 Confirmation of Stability and Activity of Ustekinumab(STELARA)

Ustekinumab is a human IgG1κ monoclonal antibody that binds with highaffinity and specificity the p40 subunit of the IL-12 and IL-23cytokines. IL-12 and IL-23 are naturally occurring cytokines that areinvolved in inflammatory and immune responses, such as natural killercell activation and CD4+ T-cell differentiation and activation. In invitro models, ustekinumab was shown to disrupt IL-12 and IL-23 mediatedsignaling and cytokine cascades by disrupting the interaction of thesecytokines with a shared cell-surface receptor chain, IL-12131. Suchactivity can be assayed, for example, using a phytohemagglutinin (PHA)blast proliferation assay and an interferon (IFN)-γ production assay(see, e.g., Oppmann et al., 2000. Novel p19 Protein Engages IL-12p40 toForm a Cytokine, IL-23, with Biological Activities Similar as Well asDistinct from IL-12 Immunity, Vol. 13, 715-725, incorporated herein byreference). Ustekinumab activity can be assayed, for example, usingphytohemagglutinin (PHA) blast proliferation and activation assays andIL-12 receptor binding assays, such as those provided below.

Phytohemagglutinin (PHA) Blast Proliferation Assay and an Interferon(IFN)-γ Production Assay

PBMC are isolated from buffy coats of healthy human donors. Humanmonocytes are obtained from PBMC, for example, by negative selectionusing Dynabeads M-450 (by Dynal A. S., Oslo. Norway). Purified monocytepopulations (85-90% CD14+) are cultured in GM-CSF (800 U/ml) and IL-4(300 U/ml) at 5×10⁵ cells/ml in RPMI+10% FBS for 6 days, with a changeof medium at day 3.

PHA blasts are derived by culture of PBMC in Yssel's medium with 0.1μg/ml PHA at 1×10⁶ cells/ml. Cells are plated at a density of 2×10⁴cells per well on a 96-well plate coated with 10 μg/ml anti-CD3 and 1μg/ml soluble anti-CD-28. Ustekinumab, incubated in containers asdescribed in the stability testing and/or stress testing methodsprovided in Example 6, is diluted to various concentrations for testing.The diluted ustekinumab samples are added to the blast cells incombination with IL-12. After 60 hours, IFNγ production is determined byELISA. To assay PHA blast proliferation, cells are then pulsed with 1μCi/well of ³H-thymidine for 6 hours, harvested, and incorporation of³H-thymidine is determined. Stimulation of IFNγ production and blastproliferation are determined for blasts contacted with ustekinumabsubject to or not subject to stress and/or stability testing to detect achange in activity of the antibody. Changes, if any, in blastproliferation or IFN-γ production as a result of storage and/or stresstesting are noted.

IL-12 Receptor Binding Assay.

Blast cells are prepared as described above. Ustekinumab, incubated incontainers as described in the stability testing and/or stress testingmethods provided in Example 6, is diluted to various concentrations fortesting. The diluted ustekinumab samples are mixed with ³H-labeled IL-12and the mixture is added to the cells. After incubation for apredetermined period of time, cells are separated from the growth media.The total amount of radioactivity in each the cells and in the growthmedia (i.e., not in the cells) is determined Inhibition of cellularuptake of IL-12 is compared for cells treated with ustekinumab subjector not subject to stress and/or stability testing. Changes, if any, inIL-12 receptor binding as a result of storage and/or stress testing arenoted.

Example 15 Confirmation of Stability and Activity of Siltuximab(CNTO-328)

Siltuximab (CNTO-328) is a chimeric, murine-human monoclonal antibodythat binds with high affinity and specificity to interleukin-6 (IL-6).There are at least two major biological functions of IL-6: mediation ofacute phase proteins, including C-reactive protein (CRP), and modulationof cell differentiation and activation. Acute phase proteins are knownto regulate immune responses, mediate inflammation, and play a role intissue remodeling. As a differentiation and activation factor, IL-6induces B cells to differentiate and secrete antibody, induces T cellsto differentiate into cytotoxic T cells, activates cell signalingfactors, and in conjunction with IL3, promotes hematopoiesis. IL-6 isprominently involved in many critical bodily functions and processes. Asa result, physiological processes including bone metabolism, neoplastictransformation, and immune and inflammatory responses can be enhanced,suppressed, or prevented by manipulation of the biological activity ofIL-6 in vivo by means of an antibody (Adebanjo et al., 1998. Mode ofAction of Interleukin-6 on Mature Osteoclasts Novel Interactions withExtracellular Ca21 Sensing in the Regulation of Osteoclastic BoneResorption. J. Cell Bio. 142:1347-1356, incorporated herein byreference). Activity of siltuximba can be determined, for example, usingan IL-6 receptor binding assay or an IL-6 induced STAT3 activation assaysuch as those provided below.

IL-6 Receptor Binding Assay

A number of human cell types express the IL-6 receptor including variousblood lymphocytes and osteoclasts. Osteoclasts are grown in cultureusing routine methods. Samples of siltuxumab is subjected or notsubjected to stress and/or stability testing using methods such as thoseprovided in Example 6 and serially diluted for testing. The dilutedsiltuxumab samples are combined with IL-6 and subsequently added toculture media. Cells are incubated for a predetermined interval, fixed,and stained for fluorescence confocal microscopy. Images are capturedand quantitated (e.g., using VOXEL) to determine the intensity ofperipheral cell membrane staining as compared to whole cell staining.The amount of inhibition of receptor internalization is compared betweenthe cells incubated with siltuxumab samples subjected or not subjectedto stress and/or stability testing to identify changes in activitylevel. Changes, if any, in IL-6 receptor binding as a result of storageand/or stress testing are noted.

IL-6 Induced STAT3 Activation Assay

IL-6 stimulates STAT3 activation in human macrophages. Activation ofSTAT3 is analyzed by monitoring its tyrosine phosphorylation in wholecell lysates by western blotting. Human monocytes are isolated frombuffy coats with a Ficoll gradient, followed by hypertonic densitycentrifugation in Percoll. After 30-min cultivation in RPMI supplementedwith 5% human serum and 1% L-glutamine, the monocytes become adherent.After attachment to the plate, monocytes are washed three times withSMEM Spinner medium to remove contaminating lymphocytes. Experiments areperformed after 4 days of cultivation. All solutions and materialscontacting monocytes/macrophages are LPS free.

Siltuxumab samples subject to or not subject to stress and/or stabilitytesting are diluted for use in STAT3 activation assays. Dilute samplesare mixed with human IL-6 and added to culture media for a predeterminedtime period.

After treatment with IL-6 and siltuxumab, cells are placed on ice. Mediais removed, cells are washed, whole cell lysates are prepared, and theconcentration of proteins in the lysates are determined. Equivalentamounts of total protein are loaded onto a gel, subject to SDS-PAGE,transferred to nitrocellulose, and probed with antibodies to each totalSTAT3 and phosphorylated STAT3. The amount of total STAT 3 andphosphorylated STAT3 signal is quantitated, e.g., using aphosphorimager. The ratio of phosphorylated STAT3 to total STAT3 iscalculated to determine changes in activity of siltuxumab as a result ofstability and/or stress testing. Changes, if any, in STAT3 activation asa result of storage and/or stress testing are noted.

Example 16 Confirmation of Stability and Activity of Golimumab (SIMPONI)

Golimumab (SIMPONI, CNTO-148) is a human IgG1κ monoclonal antibody. Itforms high affinity, stable complexes with both the soluble andtransmembrane bioactive forms of human tumour necrosis factor (TNF),which prevents the binding of TNF to its receptors. The binding of humanTNF by golimumab has been shown to neutralise TNF-induced cell surfaceexpression of the adhesion molecules E-selectin, vascular cell adhesionmolecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 by humanendothelial cells. TNF-induced secretion of interleukin (IL)-6, IL-8 andgranulocyte-macrophage colony stimulating factor (GM-CSF) by humanendothelial cells has also been shown to be inhibited by golimumab.Golimumab activity can be assessed, for example, using a TNF receptorbinding assay or a HUVEC E-selectin binding assay such as those providedbelow.

TNF Receptor Binding Assay

Human TNFα is iodinated using a commercially available kit, e.g.,Iodogen-coated tube (PIERCE). The iodinated protein is desalted e.g.,using a PD-10 column, and the pooled fractions are stored at 4° C. inPBS-0.1% bovine serum albumin.

Microtiter plates are coated with goat anti-human IgG Fc, washed andthen incubated with human TNFR1 or TNFR2 receptor fusion protein usingknown methods (see, e.g., Shealy et al., 2010. Characterization ofgolimumab, a human monoclonal antibody specific for human tumor necrosisfactor α. mAbs. 2:428-439, incorporated herein by reference). Serialdilutions of samples of golimumab subject to and not subject to stressand/or stability testing as set forth in Example 6, and anisotype-matched, negative control human IgG1 mAb antibody arepre-incubated with ¹²⁵I-TNFα (30 ng/mL) for 30 min at 37° C. Thegolimumab dilutions are added to triplicate wells and incubated for 2 hat 37° C. Plates are washed three times and the wells were counted in agamma counter. The amount of iodinated TNFα bound to the wells iscompared for samples of golimumab subject to and not subject to stressand/or stability testing. Changes, if any, in TNF receptor binding as aresult of storage and/or stress testing are noted.

HUVEC E-Selectin Binding Assay

Mouse anti-human E-selectin is iodinated using routine methods such asthose provide above. Subsequently, early passage HUVEC are thawed,washed with EGM®-2 medium (Lonza, Basel, Switzerland) and dispensed into96-well microtiter plates (5,000 cells/well). Cells are grown toconfluence at 37° C. Serial dilutions of samples of golimumab subject toor not subject to stress and/or stability testing as in Example 6, andnegative control mAb are prepared in HUVEC medium containing 1 ng/mLhuman TNFα and pre-incubated for 20 min at 37° C. The medium is removedfrom the microtiter wells seeded with cells and the golimumab sampledilutions are dispensed in duplicate. The cells are incubated at 37° C.for 4 hours and then washed with RPMI 1640 medium. Wells are thenincubated with ¹²⁵I-anti-E-selectin (0.1 mg/mL, 50 mL/well) for 1 hourat 37° C., washed three times and counted in a gamma counter. The amountof iodinated E-selectin bound to the wells is compared for samples ofgolimumab subject to and not subject to stress and/or stability testing.Changes, if any, in E-selectin binding as a result of storage and/orstress testing are noted.

Example 16 Confirmation of Stability and Activity of Fulranumab (AMG403)

Fulranumab (AMG403) is a monoclonal antibody directed against nervegrowth factor (NGF) with potential analgesic activity. Nerve growthfactor is a small secreted protein that is important for the growth,maintenance, and survival of certain target neurons and the perceptionof pain. NGF binds to high-affinity tyrosine kinase receptor TrkA which,in turn, leads to the activation of a number of downstream signalingfactors including the serine/threonine kinase, Akt, ribosomal s6 kinase(RSK), the BH3-only protein Bim, Rac1 and Cdc42, mitogen-activatedprotein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases(JNKs), and c-Jun. The activity of fulranumab subject to stress and/orstability testing as set forth in Example 6, can be characterized bydetection of inhibition of binding to TrkA either directly, or bydetection of modulation in activation of one or more of the downstreamTrkA signaling factors using routine methods such as those providedbelow.

NGF-Induced Modulation of Bim-EL Phosphorylation Assay

A, NGF acutely regulates the electrophoretic mobility of Bim-EL andbrings about long term down-regulation of Bim-EL expression. NGF isadded to various concentrations of samples of fulranumab subject or notsubject to stress and/or stability testing as set forth in Example 6.The mixtures are added to PC12 cells grown in the absence of NGF for apredetermined time (e.g., 10 minutes to 2 hours). Cell lysates areprepared and equivalent amounts of total protein are separated bySDS-PAGE and probed by Western blotting using enhanced chemiluminescencefor the expression of Bim and ERK1 using routine methods (see, e.g.,Biswas and Greene, 2002. Nerve Growth Factor (NGF) Down-regulates theBcl-2 Homology 3 (BH3) Domain-only Protein Bim and Suppresses ItsProapoptotic Activity by Phosphorylation. J. Biol. Chem.277:49511-49516, incorporated herein by reference.) NGF additionpromotes a shift in Bim-EL electrohoretic mobility as a result ofphosphorylation of Bim-EL. The change in phosphorylation of Bim-EL iscompared from the cells treated with fulranumab subject or not subjectto stress and/or stability testing. Changes, if any, in BIM-ELphosphorylation as a result of storage and/or stress testing are noted.

Dopamine Uptake Assay in Mesencephalic Neurons

Embryonic mesencephalic neurons are prepared and cultured using routinemethods (see, e.g., US Patent Publication No. 20100034818, incorporatedherein by reference). BDNF at 10 ng/ml was added to the cells 2 hoursafter plating, followed by serial concentrations of fulranumab samplessubject or not subject to stress and/or stability testing using themethods set forth in Example 6. Anti-BDNF antibody was used as apositive control.

Dopamine uptake assay are carried out as described previously (Friedmanand Mytilineou, 1987. The toxicity of MPTP to dopamine neurons inculture is reduced at high concentrations. Neurosci. Lett. 79:65-72). Atday 6, cultures are washed once with pre-warmed Krebs-Ringer's phosphatebuffer (pH 7.4) containing 5.6 mM glucose, 1.3 mM EDTA and 0.5 mMpargylin, a monoamine oxidase inhibitor. The cultures are incubated inuptake buffer containing 50 nM [³H]DA for 60 minutes at 37° C. Uptake isstopped by removing the uptake buffer, and the cultures are washed threetimes with Krebs-Ringer's phosphate buffer. Cells are lysed to release[³H]DA by adding a liquid scintillation cocktail directly to thecultures. The cell lysates are then counted for radioactivity in aliquid scintillation counter. Low affinity DA uptake is assessed byadding 0.5 mM GBR12909, a specific inhibitor of the high affinity DAuptake sites, to the uptake buffer, and subtracted from the total uptakeamount to obtained the high affinity DA uptake value. Changes, if any,in DA uptake as a result of storage and/or stress testing are noted.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the embodiments describedherein without departing from the spirit and scope of the claimedsubject matter. Thus it is intended that the specification cover themodifications and variations of the various embodiments described hereinprovided such modification and variations come within the scope of theappended claims and their equivalents.

What is claimed is:
 1. A pharmaceutical product comprising: bortezomib,ustekinumab, golimumab, siltuximab, or AMG 403 (fulranumab) and apharmaceutically acceptable excipient; contained within a glasspharmaceutical container comprising a glass composition comprising: SiO₂in an amount greater than or equal to about 72 mol. % and less than orequal to about 78 mol. %; alkaline earth oxide comprising both MgO andCaO, wherein CaO is present in an amount up to about 1.0 mol. %, and aratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) is less than or equalto 0.5; X mol. % Al₂O₃, wherein X is greater than or equal to about 5mol. % and less than or equal to about 7 mol. %; Y mol. % alkali oxide,wherein the alkali oxide comprises Na₂O in an amount greater than about8 mol. %; and a ratio of a concentration of B₂O₃ (mol. %) in the glasscontainer to (Y mol. %−X mol. %) is less than or equal to 0.3.
 2. Thepharmaceutical product of claim 1, wherein the pharmaceutical containercomprises a compressive stress greater than or equal to 150 MPa.
 3. Thepharmaceutical product of claim 1, wherein the pharmaceutical containercomprises a compressive stress greater than or equal to 250 MPa.
 4. Thepharmaceutical product of claim 1, wherein the pharmaceutical containercomprises a depth of layer greater than 30 μm.
 5. The pharmaceuticalproduct of claim 1, wherein the pharmaceutical product comprisesincreased stability, product integrity, or efficacy.
 6. Thepharmaceutical product of claim 1 wherein the glass pharmaceuticalcontainer has a compressive stress greater than or equal to 150 MPa anda depth of layer greater than 10 μm, and wherein the pharmaceuticalproduct comprises increased stability, product integrity, or efficacy.7. The pharmaceutical product of claim 1 wherein the glasspharmaceutical container is substantially free of boron, and wherein thepharmaceutical product comprises increased stability, product integrity,or efficacy.
 8. The pharmaceutical product of claim 7, wherein the glasspharmaceutical container comprises a compressive stress greater than orequal to 150 MPa and a depth of layer greater than 25 μm.
 9. Thepharmaceutical product of claim 8, wherein the glass pharmaceuticalcontainer comprises a compressive stress greater than or equal to 300MPa and a depth of layer greater than 35 μm.
 10. The pharmaceuticalproduct of claim 7, wherein said glass pharmaceutical containercomprises a substantially homogeneous inner layer.
 11. Thepharmaceutical product of claim 10, wherein said glass pharmaceuticalcontainer comprises a compressive stress greater than or equal to 150MPa and a depth of layer greater than 25 μm.
 12. The pharmaceuticalproduct of claim 1, wherein the pharmaceutical container comprises aninternal homogeneous layer.